Published OnlineFirst October 17, 2013; DOI: 10.1158/1541-7786.MCR-13-0350
Molecular
Cancer
Research
Review
The Molecular Balancing Act of p16INK4a in Cancer and Aging
Kyle M. LaPak1 and Christin E. Burd1,2
Abstract
p16INK4a, located on chromosome 9p21.3, is lost among a cluster of neighboring tumor suppressor genes.
Although it is classically known for its capacity to inhibit cyclin-dependent kinase (CDK) activity, p16INK4a is not
just a one-trick pony. Long-term p16INK4a expression pushes cells to enter senescence, an irreversible cell-cycle
arrest that precludes the growth of would-be cancer cells but also contributes to cellular aging. Importantly, loss of
p16INK4a is one of the most frequent events in human tumors and allows precancerous lesions to bypass senescence.
Therefore, precise regulation of p16INK4a is essential to tissue homeostasis, maintaining a coordinated balance
between tumor suppression and aging. This review outlines the molecular pathways critical for proper p16INK4a
regulation and emphasizes the indispensable functions of p16INK4a in cancer, aging, and human physiology that
make this gene special. Mol Cancer Res; 12(2); 167–83. 2013 AACR.
Introduction
Every day, we depend on our cells to make the right
decision—to divide or not to divide. Proliferation is essential
for tissue homeostasis, but, when deregulated, it can both
promote cancer and lead to aging. For this reason, the
decision to replicate is tightly controlled by a complex
network of cell-cycle–regulatory proteins. In the early
1990s, it was clear that the catalytic activity of cyclindependent kinases (CDKs) was required to drive cellular
division. Less obvious were the signals that regulate CDK
activity and how these became altered in neoplastic disease.
In an attempt to address this very question, Beach and
colleagues made the observation that CDK4 bound a distinct, 16-kDa protein in cells transduced with a viral oncogene (1). Biochemical characterization of this protein, later
named p16INK4a, placed it amongst the INK4-class of cellcycle inhibitors, which bind directly to CDK4 and CDK6,
blocking phosphorylation of the retinoblastoma tumor suppressor (RB) and subsequent traversal of the G1/S cell-cycle
checkpoint (Fig. 1A; refs 2, 3). In the presence of various
stressors (e.g., oncogenic signaling, DNA damage), p16INK4a
expression blocks inappropriate cellular division, and prolonged induction of p16INK4a leads to an irreversible cellcycle arrest termed "cellular senescence".
The gene encoding p16INK4a, CDKN2A, lies within the
INK4/ARF tumor suppressor locus on human chromosome
9p21.3 (Fig. 1B). CDKN2A encodes two transcripts with
alternative transcriptional start sites (4). Both transcripts share
exons 2 and 3, but are translated in different open reading
Authors' Affiliations: Departments of 1Molecular Genetics and 2Molecular
and Cellular Biochemistry, The Ohio State University, Columbus, Ohio
Corresponding Author: Christin E. Burd, Biomedical Research Tower, Rm
586, The Ohio State University, 460 W. 12th Avenue, Columbus, OH 43210.
Phone: 614-688-7569; Fax: 614-292-6356; E-mail: [email protected]
doi: 10.1158/1541-7786.MCR-13-0350
2013 American Association for Cancer Research.
frames (ORF) to yield two distinct proteins: p16INK4a and
ARF (p14ARF in humans and p19ARF in mice). In addition to
CDKN2A, the INK4/ARF locus encodes a third tumor
suppressor protein, p15INK4b, just upstream of the ARF
promoter (3). Discovered through homology-based cDNA
library screens, p15INK4b functions analogously to p16INK4a,
directly blocking the interaction of CDK4/6 with D-type
cyclins (2, 3). In contrast to p15INK4b and p16INK4a, which
function to inhibit RB phosphorylation, ARF expression
stabilizes and thereby activates another tumor suppressor,
p53 (5, 6). Like the INK family of inhibitors, p53 functions to
block inappropriate proliferation and cellular transformation.
Through a poorly understood mechanism, likely dependent
upon cell type and transcriptional output, p53 activation can
trigger either apoptosis or cell-cycle arrest (7). A fourth INK4/
ARF transcript, ANRIL (Antisense Noncoding RNA in the
INK4/ARF Locus), was recently discovered in a familial
melanoma kindred with neural system tumors (8). The
ANRIL transcript runs antisense to p15INK4b and encodes a
long, noncoding RNA elevated in prostate cancer and leukemia (9, 10). ANRIL is proposed to function as an epigenetic
regulator of INK4/ARF gene transcription, targeting histonemodifying enzymes to the locus (see the Discussion section).
In summary, the INK4/ARF locus is a relatively small (110
kb), but complex locus, essential to the proper maintenance of
cell-cycle control and tumor suppression. In this review, we
focus on the founding member of the INK4/ARF locus,
p16INK4a, and discuss what is known and unknown about
p16INK4a regulation in cancer and aging.
CDK4/6-independent roles of p16INK4a
Several lines of evidence suggest that p16INK4a may
function both through CDK4/6-dependent and -independent mechanisms to regulate the cell cycle. CYCLIN D–
CDK4/6 complexes are stabilized by interactions with the
CDK2 inhibitors, p21CIP1, p27KIP1, and p57KIP2, and serve
to titrate these proteins away from CDK2 (11–14). Subsequent expression of p16INK4a or p15INK4b causes these
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LaPak and Burd
INK4b
INK4a
Figure 1. Function, structure, and polymorphisms of the INK4/ARF locus. A, p15
and p16
both function in the RB tumor suppressor pathway through
ARF
inhibition of CDK4/6 activity. Expression of p14
inhibits the E3 ubiquitin ligase activity of MDM2, leading to stabilization of p53. The p53 and RB
pathways play integral roles in blocking inappropriate cellular proliferation. B, packed into 35 kb of chromosome 9p21.3 are three well-characterized tumor
ARF
INK4b
INK4a
suppressor genes: p14 , p15
, and p16
. GWAS have implicated 9p21.3 SNPs in cancer, heart disease, glaucoma, type 2 diabetes, autism, and
endometriosis. The majority of the SNPs lie outside of the coding regions in a recently discovered long, noncoding RNA, ANRIL. Of the
identified SNPs, those that have been shown to correlate with CDKN2A expression in at least one study are filled with gray. Other SNPs that have
not been correlated with CDKN2A expression in validation studies, or have yet to be examined are filled with black or white, respectively.
complexes to disassociate, releasing sequestered CDK2 inhibitors (15). This process, known as "CDK inhibitor reshuffling", has been documented in a growing list of cell
lines, and several lines of evidence support the biologic
relevance of this model. Mice harboring kinase-dead Cdk4
or Cyclin D1 alleles that retain p27KIP1-binding capacity
(Cyclin D1K112E, Cdk4D158N) display heightened CDK2
activity (16–18) and fewer developmental defects than
Cyclin D1 knockouts (KOs). The same observation holds
true for a Cyclin D1 knockin mutation incapable of binding
RB (Cyclin D1DLxCxE; ref. 19). As such, it is not surprising
that p27KIP1 deletion can rescue the retinal hypoplasia and
early mortality phenotypes of Cyclin D1-null mice (20, 21).
More recently, the biologic relevance of CDK inhibitor
reshuffling has come under scrutiny. Knockin mice harboring p16INK4a-insensitive Cdk4 and Cdk6 alleles still capable
of binding p27KIP1 (Cdk6R31C and Cdk4R21C, respectively)
do not display the phenotypes predicted by this model
(18, 22). The decreased p16INK4a-binding capacity of these
mutants should promote p27KIP1 sequestration and
enhanced CDK2 activity, but neither cells from the liver
or testes of Cdk4R21C mice show changes in the composition
of CDK2–Cyclin complexes, nor do thymocytes harboring
the Cdk6R31C allele (18, 22). These data suggest that, in at
least a subset of cell types, the kinase activity of CDK4/6 is
predominantly responsible for proliferative control. KO
mice lacking a single CDK4/6 inhibitor (p16INK4a, p15INK4b
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Mol Cancer Res; 12(2) February 2014
or p19INK4d) develop normally, and are born at expected
mendelian ratios (23–25). In contrast, p18INK4c KOs are
characterized by organomegaly; yet, the association of
p27KIP1 with CDK2 complexes is unchanged in these
animals (26). Work examining combined loss of p15INK4b
and p18INK4c (24) or p27KIP1 and p18INK4c (26) in mice
suggests that distinct mechanisms are used by each inhibitor
to control cellular proliferation. This result is in contrast to
the CDK inhibitor reshuffling model wherein co-deletion
would be predicted to concertedly promote CDK2 activity.
However, it is important to note that none of these publications contest the fact that CDK2 inhibitors bind
CYCLIN D–CDK4/6 complexes and are released upon
p16INK4a expression. Moreover, recent findings suggest that
p16INK4a may contribute to cell-cycle regulation through
additional CDK-independent mechanisms. Specifically,
expression of p16INK4a has been reported to stabilize
p21CIP1, and may inhibit the AUF1-dependent decay of
p21CIP1, cyclin D1, and e2f1 mRNA (27, 28). As a whole,
these data provide evidence that the cell-cycle–related functions of p16INK4a may extend beyond CDK4/6 inhibition to
include the regulation of other CDK-CYCLIN targets.
Transcriptional, Translational, and Epigenetic
Regulation of p16INK4a
To maintain tissue homeostasis and prevent cancer, the
ability of p16INK4a to inhibit cellular proliferation must be
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p16INK4a in Cancer and Aging
tightly controlled. In this section, we discuss the role of
chromatin, transcriptional cofactors, and RNAs in maintaining proper p16INK4a expression. In addition, we highlight the complexity and redundancy of p16INK4a regulatory
pathways required for proper proliferative control.
p16INK4a repression by polycomb group complexes
Chromatin modifications by the polycomb group complexes (PcG), PRC1 and PRC2, are critical to the homeostatic regulation of INK4/ARF gene expression (Fig. 2A).
The PRC2 complex is made up of four core components:
EZH1/2, EED, SUZ12, and RBAp46/48. EZH1 or -2
serves as the catalytic subunit of PRC2, and functions only
in the presence of EED and SUZ12 to compact chromatin
through the di- and trimethylation of histone H3 lysine 27
(H3K27me2/3; refs 29, 30). H3K27me3 is recognized by a
chromodomain-containing CBX protein family member
associated with PRC1. In this manner, PRC1 is recruited
to the INK4/ARF locus, where it catalyzes the ubiquitylation
of histone H2A lysine 119 (H2AK119ub), resulting in
further chromatin compaction and gene silencing (31).
Multiple variants of the PRC1 complex have been identified
in vivo, each containing homologs of the Drosophila Posterior Sex Comb (Psc; NSPC1/PCGF1, MEL-18/PCGF2,
RNF3/PCGF3,
BMI1/PCGF4,
RNF159/PCGF5,
RNF134/PCGF6), Polycomb (Pc; CBX2, CBX4, CBX6,
CBX7, CBX8), Sex Combs Extra (RING1, RING2), and
Polyhomeotic proteins (Ph; HPH1, HPH2, HPH3; ref. 32).
PRC1 complexes contain a single Psc and Pc homolog; yet,
Maertens and colleagues observed binding of MEL-18,
BMI-1, CBX7, and CBX8 to the repressed INK4/ARF locus
(32). Further investigation revealed that multiple PRC1
variants bind to the p16INK4a promoter, working in a
concerted manner to control gene expression (32). It remains
INK4a
to be determined whether recently reported noncanonical
PRC1 complexes that lack a Pc homolog contain RYBP/
YAF2, are recruited to chromatin independent of
H3K27me3 (33), and can also function to regulate INK4/
ARF gene expression. However, regardless of their composition, PRC1 and -2 complexes clearly bind throughout the
INK4/ARF locus and repress p16INK4a expression in young,
unstressed cells (31). Maintenance of this repression may be
partially dependent on the ubiquitin-specific protease,
USP11, which was also shown by Maertens and colleagues
to bind and stabilize the PRC1 complex (34). In their work,
depletion of USP11 caused polycomb complexes to dissociate from the INK4/ARF locus leading to subsequent derepression of p16INK4a.
The importance of PRC1 and PRC2 for proper p16INK4a
regulation may be best exemplified by the phenotypes of
polycomb KO mice. Deletion of the PRC1 component,
Bmi1, results in homeotic skeletal transformations and
lymphoid and neurologic defects (35). Many of these phenotypes are attributable to the deregulation of homeobox
gene expression; however, the lymphoid and neurologic
defects observed in Bmi1 KO mice can be almost completely
rescued by INK4/ARF deletion (36). Here, INK4/ARF loss
reverses the self-renewal defects of Bmi1-null hematopoietic
and neuronal progenitors (37, 38). Together, these data
show that PRC1 regulation of p16INK4a expression is
required for proper development, stem cell maintenance,
and homeostasis. In contrast to PRC1-null animals that
survive gestation, deletion of the PRC2 members, Ezh2 or
Suz12, is embryonic lethal (39, 40). For this reason, conditional KO alleles are required to assess the biologic functions of PRC2. By using such alleles to delete Ezh2 in the
brain, pancreas, and embryonic skin, phenotypic outcomes
have been observed. Specifically, loss of Ezh2 in murine
INK4a
Figure 2. Mechanisms of p16
regulation by chromatin modification. A, p16
is negatively regulated by the histone-modifying complexes, PRC1 and
PRC2, which combine to lay down repressive marks (e.g., H3K27me3, H2AK119ub) throughout the INK4/ARF locus. Several associated proteins (AP),
INK4a
including RNA-binding proteins (RBP), are reported to facilitate PRC1/2 interaction with the p16
promoter. APs and RBPs discussed in the text are shown.
Elevated expression of the PRC2 component, EZH2, is also reported to enhance INK4/ARF gene silencing. Proteins reported to transactivate EZH2, leading to
INK4a
INK4a
PRC-mediated silencing of p16
are depicted. B, activation of p16
is associated with decreases in PRC1/2 levels and the removal of repressive
INK4a
histone marks. JMJD3 demethylates H3K27me3 and subsequent chromatin decondensation promotes access by transcription factors and p16
transcription whereas JDP2 binds H3K27 and prevents further methylation. CTCF maintains chromosomal boundaries and three-dimensional structure
INK4a
of the chromatin surrounding p16
.
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pancreatic islets causes a diabetic phenotype associated with
increased b-cell expression of both p16INK4a and ARF (41).
In contrast, Ezh2 deletion in the brain and skin resulted in
only mild differentiation defects (42, 43). The recent observation that EZH1 is expressed in many adult tissues and also
catalyzes histone H3 methylation led to the hypothesis that
EZH1 may compensate for EZH2 loss in some settings.
Indeed, deletion of both Ezh1 and -2 caused severe defects in
murine skin morphogenesis associated with a >70-fold
increase in p16INK4a/ARF expression (43).
Supporting a role for EZH2 in maintaining proliferative
homeostasis, human germline mutations in EZH2 give rise
to Weaver syndrome, a congenital disorder characterized by
uncontrolled and rapid growth (44). Although this phenotype could be attributed to p16INK4a silencing, few reports
have attempted to functionally characterize the EZH2 missense mutations commonly associated with Weaver syndrome (44). Instead, work has focused on recurring point
mutations reported in B-cell lymphoma. These mutations
localize to tyrosine 641 of the EZH2 SET domain, resulting
in the production of a neomorphic protein with enhanced
H3K27 di- and trimethylation activity (45). Of importance,
not all cancer-associated EZH2 mutants are gain-of-function
alleles. In myleloid neoplasms, missense, nonsense, and
frameshift mutations in EZH2 have been described that
lack a catalytic SET domain (46). In addition, the expression
of wild-type EZH2 has been reported to cause p16INK4a
silencing in SNF5-deficient malignant rhabdoid tumors
(MRT; ref. 47). Deletion or pharmaceutical inhibition of
EZH2 activity in cells from these tumors increases p16INK4a
expression, resulting in cell-cycle inhibition (47, 48).
Together, these observations suggest that EZH2 activity
must be tightly controlled in order to maintain proliferative
homeostasis and proper p16INK4a regulation.
Recruiting polycomb to CDKN2A
In Drosophila melanogaster, polycomb group proteins are
recruited to defined DNA-binding sites termed, Polycomb
repressive elements (PRE; reviewed in ref. 49). In contrast,
few mammalian PREs have been identified to date, leading
many to speculate that other DNA-binding proteins or
RNAs must guide polycomb complexes to target genes like
the INK4/ARF locus. Recent work suggests that long, noncoding RNAs (lncRNA) can serve as scaffolds for polycomb
recruitment and epigenetic gene silencing. The discovery of
disease-linked polymorphisms within ANRIL prompted
investigation into whether this lncRNA could function in
a similar manner to regulate INK4/ARF gene transcription.
Through RNA binding assays, a study by Yap and colleagues
showed that CBX7 interacts with both ANRIL and
H3K27me3 to promote INK4/ARF gene silencing (Fig.
2A; ref. 10). Subsequently, ANRIL binding to SUZ12, a
component of the PRC2 complex, was reported to promote
silencing of p15INK4b, but not p16INK4a (50). Together with
these data, a report showing that MOV10—a putative RNA
helicase and PRC1 binding partner—is required for
p16INK4a repression, supports a role for ANRIL in polycomb
recruitment to the INK4/ARF locus (51). Whether MOV10
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Mol Cancer Res; 12(2) February 2014
binds ANRIL to facilitate PRC1 interaction with the INK4/
ARF locus has yet to be determined. However, together,
these data make a strong case for the role of ANRIL in
epigenetic silencing of p16INK4a.
ANRIL-independent mechanisms are also suggested to
recruit PcG complexes to the p16INK4a locus. Recently,
H2.0-like homeobox 1 (HLX1), a homeobox (HOX) protein,
was shown by Martin and colleagues to facilitate PRC2
recruitment to the p16INK4a promoter (Fig. 2A; ref. 52).
Although the mechanism has yet to be defined, six other
homeobox-containing proteins (HOXA9, DLX3, HOXB13,
HOXC13, HOXD3, and HOXD8) were similarly reported
to participate in p16INK4a silencing (52). Given the role of
HOX genes in developmental patterning, it is interesting to
speculate that proteins like HLX1 and HOXA9 initiate tissuespecific silencing of p16INK4a. Like HOX proteins, both
TWIST1, a basic helix–loop–helix (bHLH) transcription
factor, and KDM2B, a histone demethylase, may also facilitate polycomb-mediated silencing of the INK4/ARF locus.
Ectopic expression of TWIST1 and KDM2B can cause
cellular levels of EZH2 to rise, resulting in an increase in
PRC2 activity (53). KDM2B was further reported to function
in demethylating H3K36me2/3, a common marker for DNA
polymerase II transcription (53). Furthermore, TWIST1
appears to increase BMI1 expression (54), and subsequent
recruitment of BMI1 to the p16INK4a promoter has been
linked to interactions with phosphorylated RB (pRB) and zinc
finger domain-containing protein 277 (ZFP277; refs 55, 54).
In particular, the link between pRB and p16INK4a silencing is
intriguing, as it suggests the presence of a feedback loop
wherein cells entering the S-phase repress p16INK4a expression
(55). ZFP277-mediated recruitment of BMI1 to the
p16INK4a promoter may also be linked to the cell cycle. A
study by Negishi and colleagues showed that reductions in
ZFP277 expression caused by oxidative stress could lead to
PRC1 dissociation from the p16INK4a promoter and subsequent cell-cycle arrest (54). How these mechanisms of PRC
recruitment interplay with ANRIL remains to be established,
but, certainly, the complexity of p16INK4a silencing is indicative of the importance of this gene in maintaining tissue
homeostasis.
Reversing polycomb silencing of the INK4/ARF locus
In order for normal cells to traverse the G1–S checkpoint,
p16INK4a must be maintained in a repressed state. At the
same time, induction of p16INK4a expression in the presence
of stress signals is required to prevent inappropriate cell-cycle
progression. Linking proliferative control to epigenetic regulation of the p16INK4a promoter, Bracken and colleagues
first demonstrated that RB phosphorylation during the G1 to
S-phase transition releases E2F1 to transactivate EZH2 and
EED (56). Later, these data were confirmed by an independent group who showed that p53 activation represses EZH2
expression through RB-mediated inhibition of E2F1 activity
(57). Although these results explain how the activity of
PRC2 might be curbed in the presence of stress, they do
not explain how epigenetic silencing of the p16INK4a promoter is reversed. One mechanism of removing repressive
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p16INK4a in Cancer and Aging
histone marks is via the activity of histone demethylases. In
response to oncogenic stressors, levels of the H3K27me3
demethylase, Jmjd3, increase, removing repressive histone
marks from the p16INK4a promoter (Fig. 2B; refs 58, 59).
Following histone demethylation, researchers at the
Yokoyama laboratory showed that Jun dimerization protein
2 (JDP2) may help maintain p16INK4a in an active state by
binding and sequestering H3K27 away from the actions of
PRC2 (60). These findings suggest that the activity of JDP2
and JMJD3 establishes a permissive state for p16INK4a
expression. Furthermore, unmethylated H3K27 is no longer
recognized by PRC1, and chromatin surrounding the
p16INK4a promoter is decondensed. Based upon this activity,
it is not surprising that JMJD3, like p16INK4a, serves as a
barrier to induced pluripotency (See the section "p16INK4a as
a Barrier to Pluripotency"; refs 61, 62). Clearly, interplay
among PRC1, PRC2, and histone demethylases is required
for homeostatic regulation of the p16INK4a promoter, yet,
how this crucial balance is maintained is still in question.
Moreover, it is possible that other histone demethylases,
such as lysine-specific demethylase 6A (KDM6A/UTX),
may also play a role in p16INK4a regulation.
In Drosophila, the SWI–SNF chromatin remodeling complex serves as a trithorax group activator, opposing the actions
of polycomb-mediated silencing. Similarly, SWI–SNF functions to counteract PcG silencing of p16INK4a in mammals.
Cancer cell lines deficient in the SWI–SNF component,
SNF5, induce high levels of p16INK4a upon restoration of
SNF5 expression (63). In untransformed cell lines, SNF5
binds and directly inhibits the transcription of EZH2, resulting in decreased polycomb occupancy at the p16INK4a promoter (47). Tumor cells lacking SNF5 overexpress EZH2 and
are dependent upon the activity of PRC2 to silence p16INK4a
expression and drive proliferation (47, 64).
INK4a
Chromatin regulation of p16INK4a by nonpolycomb
proteins
Epigenetic modification of the INK4/ARF locus extends
beyond polycomb-mediated silencing. The well-conserved
genomic insulator, CCCTC-motif binding factor (CTCF),
binds throughout the INK4/ARF locus (65, 66) and functions to regulate both chromatin compaction and gene
expression (Fig. 2B). Witcher and colleagues first reported
interaction of CTCF with a chromosomal boundary approximately 2 kb upstream of the p16INK4a promoter (65). In
their studies of cancer cell lines, knockdown of CTCF
resulted in the spread of heterochromatin DNA into the
INK4/ARF locus, leading to epigenetic silencing of p16INK4a
(65). In contrast to this model, Hirosue and colleagues
recently reported that decreases in CTCF expression associated with oncogene-induced senescence and promotes
decondensation of the INK4/ARF locus leading to heightened levels of p16INK4a mRNA (66). Reconciliation of these
two results is possible as rapid p16INK4a induction following
CTCF knockdown would place enormous pressure on
would-be cancer cells to epigenetically silence the INK4/
ARF locus. In fact, the observation that epigenetic silencing
of p16INK4a in breast cancers is accompanied by CTCF
disassociation from the locus (65) is consistent with a role for
CTCF in proper epigenetic regulation of INK4/ARF.
Transcriptional activators and repressors of p16INK4a
Opposing transcriptional regulators. The presence of a
permissive chromatin state alone is insufficient for p16INK4a
expression. Binding of activation factors and the subsequent
recruitment of RNA polymerase is required to initiate
p16INK4a transcription (Fig. 3). Similar to the interplay
between PRC2 and JMJD3, transcriptional regulation of
the p16INK4a promoter is tightly controlled by antagonistic
INK4a
Figure 3. Transcriptional regulation of p16
. Expression of p16
requires the action of transcription factors (green) that recruit and/or facilitate RNA
INK4a
promoter are
polymerase association with the promoter. Opposing this action are transcriptional repressors (red). Direct interactions with the p16
depicted by a solid line and indirect interactions with a dotted line. The numbers below each binding site indicate the position of protein interaction relative to
INK4a
the p16
transcriptional start site. All locations correspond to the human genome unless designated by an "(m)," which signifies the mouse
genome. Proteins predicted to share a common binding site are depicted on top of one another.
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pathways. Serving as a classic example, induction of p16INK4a
by the E-box binding transcription factors E-26 transformation-specific 1 (ETS1), E-26 transformation-specific 2
(ETS2) and E47, is directly opposed by Inhibitor of DNA
Binding 1 (ID1; ref. 67). In response to oncogenic and
senescent signaling, ETS1, ETS2, and/or E47 bind to E-box
motifs (CANNTG) within the p16INK4a promoter to stimulate gene expression (67, 68). This action is directly
antagonized by ID1, which prevents the interaction of
ETS1, ETS2, and E47 with the p16INK4a promoter (67,
68). As such, it is not surprising that Id1-null MEFs undergo
premature senescence in culture (69) and that age-related
increases in p16INK4a expression correlate directly with ets-1
levels in mice and rats (70).
Similar to ID1, TWIST1 is reported to oppose transcriptional activation of p16INK4a. The relationship between ID1
and TWIST1 was initially suggested in studies examining
the progression of benign human nevi to melanoma. In
general, nevi are nonproliferative and express elevated levels
of p16INK4a; yet, upon progression to melanoma, these
lesions frequently silence p16INK4a (71). Analysis of
TWIST1 and p16INK4a expression in nevi and melanomas
revealed an inverse correlation between these two proteins,
putting forth the hypothesis that they function in antagonistic pathways (72). Preliminary work suggested that this
antagonism might be mediated through direct interaction of
TWIST1 with ETS2 (72), however, in a recent publication
by Cakouros and colleagues, TWIST1 was reported to
inhibit p16INK4a transcription by decreasing E47 expression
(73). Clearly, further work is required to fully understand the
physiological relationship between p16INK4a and TWIST1.
In addition, it will be of interest to assess the potential role of
p16INK4a in classical TWIST1 pathways including epithelial
to mesenchymal transition, stem cell maintenance, and
tumor metastasis.
In line with the relationship among TWIST, ID1, and Ebox binding transcription factors, antagonistic interplay has
also been described between members of the Activator
Protein-1 (AP-1) family of transcription factors, c-JUN and
JUNB. Although JUNB serves to activate p16INK4a transcription by binding to three identified AP1-like sites within
the p16INK4a promoter (74), c-JUN limits p16INK4a expression (75). Certainly, a delicate balance between inhibitory
(ID1, TWIST, c-JUN), and stimulatory (JUNB, ETS, E47)
pathways is required for proper regulation of p16INK4a.
Upsetting this balance through the overexpression of inhibitors or repression of p16INK4a activators promotes the
bypass of senescence and can lead to cancer (72, 73, 76–78).
The HOX family of proteins could also be viewed as
antagonistic p16INK4a regulators. Earlier, we discussed the
potential role of HLX1, HOXA9, DLX3, HOXB13,
HOXC13, HOXD3, and HOXD8 in polycomb-mediated
repression of the INK4/ARF locus. In contrast, the HOX
proteins, VENTX, MEOX1, and MEOX2 have each been
reported to bind the p16INK4a promoter and activate gene
transcription (79–81). This proposed interplay between
HOX genes and p16INK4a regulation is suggestive of a role
for CDK4/6 inhibition in embryonic development. In spite
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Mol Cancer Res; 12(2) February 2014
of this, developmental defects are not observed in p16INK4a
KO mice or melanoma-prone kindreds harboring germline
p16INK4a deficiencies (25, 82). Whether compensatory
mechanisms are required to combat the functional loss of
p16INK4a during development is still to be determined.
Role of acetyltransferases and deacetylases in p16INK4a
regulation. Histone acetylation facilitates chromatin
decondensation and subsequent gene transactivation. As
such, transcriptional coactivators often harbor or recruit
histone acetyltransferase (HAT) activity to target gene promoters. In a pair of recent publications, Wang and colleagues
describe how the transcription factors, SP1 and HMG boxcontaining protein 1 (HBP1), recruit p300, a well-known
HAT, to the p16INK4a promoter (83, 84). In their studies,
acetylation of local chromatin as well as HBP1 promoted
decondensation of the p16INK4a promoter and subsequent
gene transactivation. However, numerous targets of p300
acetylation have been identified to date, including B-MYB, a
putative repressor of p16INK4a transcription (83, 85, 86).
Therefore, the p300–p16INK4a relationship is likely complex
and may be dependent upon the available pool of transcriptional cofactors within a given cell type.
HAT activity is opposed by histone deacetylases (HDAC),
which promote transcriptional silencing. In human cell lines,
HDACs 1 to 4 have all been reported to bind and repress
transcription of the p16INK4a promoter (52, 87–89). Most of
these interactions have been linked to bridging transcription
factors such as Lymphoid Specific Helicase (LSH), HLX1,
and ZBP-89 (87, 89), albeit one report suggested that
HDAC2 may directly bind the p16INK4a promoter (88).
Although loss of Hdac1, 2, 3, or 4 causes developmental
abnormalities and lethality in mice, none of these phenotypes have been attributed to defects in p16INK4a regulation
(90).
Age-related signaling pathways influence p16INK4a
expression. The observation that p16INK4a levels are high
in senescent cells has prompted several investigations into
the connection between prosenescent signaling and
p16INK4a regulation. For example, age-related metabolic
pathologies have long been associated with activation of the
peroxisome proliferator-activated receptors (PPARs). It is
now known that these nuclear receptors directly bind and
activate the p16INK4a promoter leading to subsequent cellcycle arrest (91, 92). Similarly, alterations in TGF-b signaling have been linked to a variety of age-related diseases
including cancer, osteoarthritis, cardiovascular disease, and
Alzheimer’s (93). Work by researchers at the Conboy laboratory has demonstrated that elevated TGF-b signaling
reduces the capacity of muscle stem cells to regenerate (94).
Here, phospho-SMAD3 has been shown to directly bind the
p16INK4a promoter, stimulating gene transcription and cellcycle arrest (95). Due to cross-talk between the TGF-b and
b-catenin/Wnt signaling pathways, it is not surprising that
aberrant Wnt signaling is also associated with age-related
disease (95). b-catenin can directly bind and activate the
p16INK4a promoter in both human and murine cells (96–
98), and recent evidence links the induction of p16INK4a by
reactive oxygen species (ROS) to b-catenin/Wnt signaling
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(99). Together, these findings suggest a strong association
between age-promoting, "gerontogenic" signals and
p16INK4a expression.
Cellular structure and p16INK4a expression
An emerging theme in p16INK4a regulation is the
potential for cytoskeletal rearrangements to influence
INK4/ARF gene transcription. In an siRNA screen of
>20,000 genes, Bishop and colleagues recently identified
GLI2, a member of the Hedgehog signaling pathway, as
an activator of p16INK4a expression (100). Interestingly,
GLI2 partially localizes to a nonmotile cytoskeletal protrusion called the primary cilium, and cultured human
mammary epithelial cells with a primary cilium expressed
lower levels of p16INK4a than those without (100). Upon
ablation of p16INK4a, the number of cells with a primary
cilium increased, suggesting a link between cellular structure and p16INK4a expression (100). Supporting this
observation, the ACTIN-nucleating enzymes ARP2 and
3 have been implicated in a second connection between
cytoskeletal structure and Ink4/Arf gene transcription.
Here, the generation of stable ARP2/3 knockdown cells
required concomitant Ink4/Arf deletion (101). These data
suggested that lamellipodial dysfunction triggers growthinhibitory Ink4/Arf gene transcription; however, the specific role of p16INK4a in this process is yet to be examined.
The role of miRNAs and RNA-binding proteins in
p16INK4a regulation
Although literature defining transcriptional and epigenetic
regulators of p16INK4a is extensive, far fewer studies have
examined posttranscriptional mechanisms of p16INK4a regulation. Discovered in an unbiased search for miRNAs silenced
during senescence (102), two independent groups have
reported that miR-24 binds and inhibits p16INK4a translation
(102, 103). Consistent with this observation, antagonists of
miR-24 cause a moderate decrease in the proliferation of
normal human keratinocytes (103). In a similar manner,
knockdown of miR-31 has been proposed to regulate cellcycle progression in murine embryo fibroblasts with altered
nuclear structure (104). Here, loss of Lamin B1 was associated
with increased miR-31 expression and p16INK4a instability
(104). This work suggests another possible connection
between cellular structure and p16INK4a regulation, linking
changes in nuclear integrity to p16INK4a expression. Together,
the associations among miR-31, miR-24, and p16INK4a
suggest that miRNAs function to control proliferation via
interactions with p16INK4a mRNA; however, it is important to
note that other cell-cycle regulators have been identified as
miR-24 targets (e.g., cdk4, cyclin A2, cyclin B1, myc, e2f2,
p14ARF, and p27KIP1; refs 103, 105, 106) and miR-31 (e.g.,
ets1, cdk1; ref. 107). Therefore, cell-cycle defects caused by
perturbations in miRNA expression likely reflect the outcome
of both p16INK4a-dependent and -independent pathways.
The let-7 family of miRNAs has also been implicated in
p16INK4a regulation, proliferative control, and stem cell
aging. The Morrison group first reported that murine let7b expression increased with age in neuronal stem cells
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(108). Although let-7b did not bind p16INK4a mRNA
directly, overexpression of let-7b reduced the expression of
High-Mobility Group AT-Hook 2 (HMGA2), causing
p16INK4a levels to increase (108). In addition to hmga2, a
growing number of RNAs involved in growth and proliferative control have been identified as let-7b targets, and,
therefore, it is not surprising that the levels of most let-7
family members decrease during tumor progression (109).
In addition to miRNAs, RNA-binding proteins can also
regulate p16INK4a translation. Interaction of the Hu RNAbinding protein, HuR, with the p16INK4a 30 UTR have been
reported to destabilize the transcript in an miRNA-independent fashion (110). Work by Zhang et al. suggests that
this action is opposed by the tRNA methyltransferase,
NSUN2, which methylates the 30 -untranslated region
(UTR) of p16INK4a to prevent HuR binding and subsequent
mRNA degradation (111). In this way, interplay between
HuR and NSUN2 may tightly control p16INK4a translation.
For example, in the presence of oxidative stress, NSUN2
levels appear to increase, tipping the balance toward mRNA
stability, p16INK4a expression, and subsequent cell-cycle
arrest (111).
The Function and Significance of p16INK4a
Expression in Cancer
In the absence of p16INK4a, both mice and humans are
predisposed to cancer (25, 82). Loss of p16INK4a function
can occur through gene deletion, methylation, or mutation,
and, therefore, comprehensive genetic analyses are required
to determine the frequency of p16INK4a silencing in cancer.
Adding to the challenge of assessing p16INK4a status in
human tumors, few studies distinguish between the two
CDKN2A transcripts: p16INK4a and ARF. Here, we compiled data from The Cancer Genome Atlas (TCGA) to reveal
that functional inactivation of CDKN2A is a frequent event
in most tumor types (Fig. 4; refs 112–118). We probed 16
tumor types to determine the percentage of cases wherein
mutational and/or copy-number changes disrupted genes
critical to the p16INK4a tumor suppressor pathway (i.e., RB1,
CDKN2A, CCND1 (CYCLIN D1), CCND2 (CYCLIN
D2), CCND3 (CYCLIN D3), CCNE1 (CYCLIN E1), and
CCNE2 (CYCLIN E2)). Tumor types displaying a high
percentage of p16INK4a tumor suppressor pathway alterations included urothelial carcinoma (BLUCA; 77%), glioblastoma multiforme (GBM; 77%), head and neck squamous cell carcinoma (HNSC; 63%), lung squamous cell
carcinoma (LUSC; 58%), and cutaneous melanoma
(SKCM; 58%; Fig. 4A). Examination of individual tumor
profiles revealed infrequent overlap between CDKN2A and
RB1 deletion, implying that these are often mutually exclusive tumorigenic events (Fig. 4B). In support of these data,
an inverse correlation between p16INK4a and RB inactivation
was previously described in lung cancer cell lines (119) and
human tumors (120). In contrast to RB, we frequently noted
alterations within other components of the p16INK4a tumor
suppressor pathway (i.e., Cyclin amplification) in CDKN2A
mutant tumors from the TCGA dataset (Fig. 4B). However,
in cases where the p16INK4a tumor suppressor pathway
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INK4a
Figure 4. Alterations in the p16
tumor suppressor pathway are frequent in human cancer. A, data from TCGA was obtained and analyzed using cBioPortal
(112, 113). The tumors analyzed are as follows: bladder urothelial carcinoma (BLUCA), glioblastoma multiforme (GBM), head and neck squamous cell
carcinoma (HNSC), lung squamous cell carcinoma (LUSC), skin cutaneous melanoma (SKCM), ovarian serous cystadenocarcinoma (OV), lung
adenocarcinoma (LUAD), stomach adenocarcinoma (STAD), breast invasive cancer (BRCA), uterine corpus endometrial carcinoma (UCEC), brain lower grade
glioma (LGG), colon and rectum adenocarcinoma (COAD/READ), prostate adenocarcinoma (PRAD), kidney renal papillary cell carcinoma (KIRP), kidney
INK4a
renal clear cell carcinoma (KIRC), acute myeloid leukemia (AML). The percentage of tumors with mutations or copy-number changes in the p16
INK4a
INK4a
tumor suppressor pathway are shown. For the purposes of this analysis, the p16
tumor suppressor pathway was defined to contain: CDKN2A (p16
),
RB1 (RB), CCND1 (CYCLIN D1), CCND2 (CYCLIN D2), CCND3 (CYCLIN D3), CCNE1 (CYCLIN E1), and CCNE2 (CYCLIN E2). B, OncoPrints from
cBioPortal show aberrations in individual tumors across the x-axis. C, methylation of CDKN2A (black bars) and RB1 (gray bars) was quantified using
HM27 or HM450 TCGA data. Genes were considered methylated if b-values exceeded 0.2.
appeared genetically intact, high levels of RB1 or CDKN2A
methylation were observed [e.g., kidney cancers (KIRP,
KIRC), colorectal adenocarcinomas (COAD/READ),
low-grade glioma (LGG), and acute myeloid leukemia
(AML); Fig. 4C]. Combining these genetic and epigenetic
data suggests that functional inactivation of the p16INK4a–
RB axis approaches 100% in many cancer types. A prior
study by Schutte and colleagues supports this claim, demonstrating inactivation of the p16INK4a–RB-axis in 49 of 50
pancreatic carcinomas (121). However, further comprehensive assessment of p16INK4a functionality in a wide variety of
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Mol Cancer Res; 12(2) February 2014
tumors will require thorough experimental and bioinformatic analyses.
p16INK4a expression in tumors
In most tumor types, p16INK4a inactivation occurs early
in tumorigenesis. For example, pancreatic intraepithelial
neoplasias frequently inactivate p16INK4a upon progression to invasive disease (122). Pressure to silence p16INK4a
presumably stems from oncogenic engagement of the RB
tumor suppressor pathway. Therefore, tumors with early
defects in RB signaling continue to express p16INK4a
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independent of progression (reviewed in ref. 123). Intense
activation of the p16INK4a promoter is believed to represent a futile attempt by the cell to curb oncogenic
proliferation in the absence of a functional G1–S checkpoint. In the clinic, immunohisotchemical p16INK4a staining is used to identify cervical and head and neck tumors
driven by oncogenic human papilloma virus (HPV) infection. Here, the HPV viral oncoprotein, E7, functionally
inactivates RB leading to increases in p16INK4a expression
(124). Although it is believed that RB inactivation is
requisite for the elevation of p16INK4a expression in
cancer, aberrations in the RB pathway are not obvious
in every tumor. The RB checkpoint is deregulated by
multiple mechanisms independent of RB1 mutation,
deletion, or methylation. Viral oncogene expression and
cyclin gene amplification represent just two possible forms
of alternative RB inactivation. Unfortunately, there is a
paucity of studies that comprehensively examine the status
of the RB pathway in multiple human tumor types.
Therefore, the critical role of this p16INK4a-induced
checkpoint may be underestimated.
Limited analyses of the RB pathway in human tumors
have revealed that p16INK4a expression can be predictive of
both tumor subtype and therapeutic response. Elevated
p16INK4a levels discern small-cell lung cancer from lung
adenocarcinoma (119, 125) and characterize the basal-like
breast cancer subtype (126). Given that tumor subtypes
often show distinct therapeutic response profiles, it is not
surprising that p16INK4a expression can predict therapeutic
efficacy. For example, elevated p16INK4a levels predict a
higher initial response to radiation therapy in prostatic
adenocarcinoma (127). However, these same p16INK4a
positive tumors are the most likely to fail androgen-deprivation therapy (128). In oropharyngeal cancer, p16INK4a
serves as a marker of oncogenic HPV infection and is
predictive of improved therapeutic response and patient
survival (129). Although p16INK4a expression clearly serves
as a relevant clinical marker, combined analysis of multiple
RB pathway members may be of additional value. Likewise,
recent data suggests that the localization of p16INK4a staining
within a tumor may provide further clinical insight (see
further sections).
The significance of stromal p16INK4a expression
Although a myriad of signals are linked to p16INK4a
regulation, most of these are triggered by intrinsic, cellautonomous events. Employing a recently developed luciferase reporter mouse, p16LUC, we have observed a second,
cell-nonautonomous mechanism of p16INK4a activation
(130). The p16LUC allele expresses firefly luciferase from
the endogenous p16INK4a promoter, allowing investigators
to dynamically track p16INK4a transcription in live animals.
When the p16LUC allele was crossed with genetically engineered mouse models (GEMM) of human cancer, a luminescent signal appeared only in the location of future tumor
formation (130). These luminescent foci were visible weeks
before tumors could be visualized or palpated, providing a
significant detection advantage over traditional monitoring
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methods. This incredible sensitivity, along with the observation that tumors maintained luciferase activity during
progression, led to the hypothesis that p16INK4a transcription is activated in the surrounding tumor stroma. Indeed,
this was the case as syngeneic transplantation of six p16LUCnegative tumor cell lines harboring a wide variety of oncogenic drivers caused stromal induction of p16LUC (Fig. 5A
and (130)). Although the specific stromal cell types responsible for this observation are yet to be identified, bone
marrow transplantation studies suggest that the p16LUC
signal originates in part from bone marrow–derived cells.
Therefore, it appears that alterations in the milieu surrounding a growing neoplasm can promote the expression of
p16INK4a in a cell-nonautonomous fashion (Fig. 5B).
Mounting evidence supports a role for stromal p16INK4a
expression in tumor initiation and progression. Fibroblasts
INK4a
Figure 5. Extrinsic versus intrinsic activation of p16
. A, injection of
Lkb1-null endometrial cancer cells into a syngenic mouse heterozygous
LUC
for the p16
reporter causes stromal luciferase expression upon tumor
growth. Injection of Matrigel vehicle on the opposite flank does not alter
INK4a
p16
expression. (See also ref. 130.) B, intrinsic signals induces
INK4a
p16
expression in damaged, senescent, or transformed cells.
Alterations in surrounding the cellular milieu can trigger the induction of
INK4a
in nearby undamaged cells through an unknown pathway.
p16
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ectopically expressing p16INK4a have altered cellular metabolism and overproduce high-energy mitochondrial fuels such
as L-lactate (131). In xenograft studies, co-injection of these
fibroblasts with MDA-MB-231 breast cancer cells increased
tumor size 2 fold, suggesting that altered stromal metabolism
caused by p16INK4a expression promotes tumor growth
(131). In support of this observation, elevated p16INK4a
levels in the stroma of human mammary ductal carcinoma in
situ (DCIS) lesions are predictive of disease recurrence
independent of other histopathologic markers such as ER
positivity (132). It remains to be determined whether the
promotion of tumorigenesis by p16INK4a-positive stromal
cells is solely a reflection of altered local metabolism. Studies
from the p16LUC mouse model suggest that p16INK4a induction in tumor-infiltrating immune cells may also promote
cancer progression by dampening the antitumor response.
Regardless of the mechanism, these data make it clear that
p16INK4a expression in the tumor stroma should not be
ignored.
Therapeutic mimicry of p16INK4a
Pharmaceutical efforts to mimic the function of
p16INK4a arose after the antitumor effects of flavopiridol
were linked to CDK inhibition (reviewed in ref. 133).
Flavopiridol (alvocidib) was originally touted as an inhibitor of EGF receptor (EGFR), but later was shown to
inhibit the growth of a wide range of cancer cells at an
IC50 much lower than that required to block EGFR
activation. Broad inhibition of CDKs, including CDK4
(IC50 < 120 nmol/L), was later identified as the mechanism
of flavopiridol action (134). After several promising phase I
trials, flavopiridol failed in phase II, showing significant
activity only in cases of relapsed chronic lymphocytic
leukemia (135). Based upon these disappointing results,
Sanofi-Aventis halted production of flavopiridol in 2010.
For other nonselective CDK inhibitors, multiple toxicities
and poor solubility prevented successful transition to the
clinic. However, recent efforts to generate potent and
selective CDK inhibitors appear more fruitful.
Of particular interest to the pharmaceutical industry,
selective CDK4/6 inhibitors have shown promise in early
clinical trials (reviewed in ref. 136). Knowledge of p16INK4a
regulation and function has bolstered these efforts by providing biomarkers of therapeutic efficacy and identifying
patient populations wherein response is likely. Often, clinical trials of CDK4/6 inhibitors now exclude RB-null
tumors, which are naturally resistant to the effects of ectopic
p16INK4a expression. In addition, the observation that
p16INK4a is markedly upregulated in response to RB-inactivating viral oncoproteins (e.g., E7, T-Antigen) has prompted
the exclusion of tumors expressing high levels of p16INK4a.
Finally, studies of CDK KO mice are assisting these
efforts, identifying cell and tumor types that are more
reliant upon the activity of CDK4/6 to drive proliferation
than that of CDK2. Employing this knowledge, CDK4/6
inhibitors are showing efficacy, and a number of drugs
have now entered phase II and III clinical trials [phase II:
LEE011(Novartis) and Ly2835219(Eli Lilly); phase III:
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MK-7965/dinaciclib (Merck) and PD-0332991/palbociclib (Pfizer); ref. 137).
p16INK4a selectively binds CDK4 and -6 in vitro (2);
however, achieving such therapeutic specificity is more
challenging. In fact, Merck attributes the success of dinaciclib in chronic lymphocytic leukemia to inhibition of
CDK9, not CDK4/6 (137). Moreover, the observation that
p16INK4a may contribute to CDK4/6-independent cellcycle regulation (i.e., through CDK inhibitor reshuffling)
suggests that pharmaceutical CDK4/6 inhibitors may not
fully recapitulate the potency of p16INK4a. Although adverse
effects associated with CDK4/6-selective inhibitors are mild
(i.e., limited bone marrow suppression; ref. 136), concerns
about the long-term efficacy of such therapeutics is increasing. In particular, mechanisms to bypass the requirement for
CDK4/6 are already known, including RB loss and increased
CDK2 activity. How quickly tumors will exploit these
pathways to subvert therapeutic treatment is unknown.
Initial trials of PD-0332991 in mantle cell lymphomas speak
to this concern. Although 89% of study participants showed
reduced phospho-RB staining at 3 weeks, only 18% of
tumors responded to therapy, suggesting that resistance was
rapidly acquired (138). In addition to concerns about
resistance, the role of p16INK4a expression in the tumor
stroma remains undefined, and inhibition of the local
immune response or altered cellular metabolism may function to promote growth and metastasis. In fact, fibroblasts
exposed to PD-0332991 adapt a tumor-promoting metabolic phenotype similar to that of fibroblasts overexpressing
p16INK4a (131). Therefore, therapeutic mimetics could have
similar, detrimental consequences on the tumor microenvironment. A final concern is the long-term effects of
CDK4/6 inhibition on cellular senescence. Prolonged
expression of p16INK4a promotes senescence and decreases
regenerative capacity (see following sections). In a similar
manner, CKD4/6 inhibitors may influence tissue aging.
However, under normal physiologic conditions, stem cell
populations are, by and large, quiescent and, thus, unlikely
to be altered by CDK4/6 inhibitors. It may be that stress
and/or mitogenic signaling, which often accompany
p16INK4a induction in vitro, is required to elicit senescence,
and, therefore, the effects of CDK4/6 inhibition on aging
will be minimal. As more than half of all cancers are
diagnosed in people 65 years and older, determining whether
CDK4/6 therapeutics exacerbate age-related disease will be
of significant interest.
The Role of p16INK4a in Senescence and Aging
p16INK4a marks biologic senescence
As noted first by Sherr and colleagues (139), p16INK4a
levels increase with aging. In fact, detailed quantification has
demonstrated a direct increase in p16INK4a expression with
chronologic age in all mammalian species tested to date (140).
Such increases in p16INK4a are exponential, increasing
approximately 16-fold during the average human lifespan
and making p16INK4a one of the most robust aging biomarkers characterized to date (141). The induction of senescence
and p16INK4a expression is traditionally associated with a wide
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variety of intrinsic cellular stressors including: DNA damage,
telomere erosion, ROS, and stalled replication forks (reviewed
in ref. 142). However, use of the p16LUC reporter mouse has
provided evidence that undefined, extrinsic signals can also
trigger p16INK4a transcription in a cell nonautonomous
fashion (130). Using this same reporter to compare the
dynamics of p16INK4a expression in mice with that observed
in humans showed a direct correlation between the rate of
p16INK4a accumulation and lifespan (130). Furthermore, data
from progeroid and calorically restricted rodents suggest that
p16INK4a serves as a marker of biologic, rather than chronologic aging (70, 143, 144). Supporting this observation in
humans, smoking and chemotherapy are associated with
elevated p16INK4a expression in the human population
(141, 145). Moreover, skin biopsies from long-lived nonagenarian cohorts have fewer p16INK4a-positive cells than their
age-matched partners (146). Clinically, use of p16INK4a as a
marker of biologic aging could provide quantitative measures
of patient fitness including immune function and chemotherapeutic tolerance (145, 147). Assessment of p16INK4a
levels prior to organ transplantation may also aid in the
identification of biologically "younger" donor tissues with
increased potential for success (148–150).
A causal role for p16INK4a in aging
Several lines of evidence suggest that p16INK4a is not only a
biomarker of aging, but also causes aging in many cell types.
Using p16INK4a transgenic and KO mice, the cell-autonomous role of p16INK4a in aging has been investigated. In
murine hematopoietic stem cells, T cells, pancreatic b-cells,
and neural progenitors of the subventricular zone, agerelated p16INK4a expression causes a decline in regenerative
capacity (151–154). A caveat to these initial studies was the
use of germline KO mice; however, strategies employing
conditional p16INK4a loss or siRNAs have since confirmed
these findings in T cells and pancreatic b-cells (154, 155).
Supporting the idea that tissues expressing less p16INK4a are
more biologically fit, kidney transplant success is higher in
p16INK4a-low donor organs (148–150). These data put forth
the hypothesis that p16INK4a expression drives cellular
senescence, resulting in decreased regenerative potential. As
a proof of principal, work from the Van Deursen laboratory
showed that deletion of p16INK4a-expressing cells from a
BubR1-deficient, progeroid mouse model reduced many
aging phenotypes (e.g., sarcopenia, cataracts, loss of adiposity; ref. 144). Further work by this group showed that both
muscle and adipocyte progenitors from these mice express
high levels of p16INK4a, suggesting that even the deletion of
senescent stem cells can promote improved regenerative
capacity (156). Unfortunately, these animals did not live
longer, owing to the development of cardiac pathologies, and
a similar experiment has yet to be reported in wild-type mice
(144). One explanation for why these animals were not longlived is that the gerontogenic effects of p16INK4a expression
are tissue specific. Indeed, p16INK4a levels do not seem to
influence the regenerative capacity of murine melanocyte
stem cells or neuronal progenitors of the dentate gyrus
(unpublished observations and refs 153, 157). Other
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mechanisms of curbing age-related p16INK4a induction have
been reported in mice. For example, activation of plateletderived growth factor receptor (PDGFR) signaling in elderly
murine pancreatic b-cells increased the regenerative capacity
of these cells via repression of p16INK4a expression (158).
Likewise, administration of fibroblast growth factor 7
(FGF7) reduced p16INK4a levels and increased the number
of early T-cell progenitors in 15- to 18-month-old mice,
thereby partially rescuing the known decline in thymopoiesis
with age (159). Apart from these studies, analyses of
p16INK4a-mediated regenerative decline are limited to a
small number of tissues; therefore, the potential outcome of
p16INK4a-directed therapies remains uncertain. Clearly, further characterization of the relationship among p16INK4a
expression, senescence, and regenerative capacity in vivo
would have broad implications for the therapeutic treatment
of age-related disease.
The role of p16INK4a in intrinsic versus extrinsic aging
Senescence is typically viewed as a response to detrimental,
intrinsic cellular events; however, recent evidence suggests
that extrinsic signals also contribute to tissue aging. Earlier,
we discussed the potential for neoplastic transformation to
initiate cell nonautonomous p16INK4a expression. Although
the extracellular mediators of stromal p16INK4a induction are
undefined, several signaling pathways have been linked to
the expression of p16INK4a in aging tissues. In muscle
satellite cells, age-associated increases in local TGF-b
production activate SMAD3, which in turn binds to the
p16INK4a promoter to initiate gene transcription (94).
NOTCH signaling antagonizes TGF-b, and, in doing so,
can alleviate age-related declines in satellite cell function
(94). Similar to TGF-b signaling, changes in thymic and
bone marrow structure are reported to promote aging in
local progenitor cell populations (142). Together, these
findings put forth the model of "niche aging," wherein
stromal changes influence the regenerative capacity of
local progenitors. However, parsing the role of the senescent cell versus the niche in aging biology becomes
somewhat of a chicken and egg question. After all, senescent cells themselves secrete a large number of proinflammatory cytokines associated with age-related disease
(reviewed in ref. 160). Future studies aimed at identifying
cell nonautonomous p16INK4a activation signals are clearly
needed to better understand the induction of senescence
during physiologic aging.
p16INK4a as a barrier to pluripotency
Work with induced pluripotent stem cells (iPS) also
implicates p16INK4a as a modulator of regenerative capacity.
During iPS generation, senescence induced by the fourfactor cocktail of OCT4, SOX2, KLF4, and c-MYC serves as
a barrier to efficient reprogramming (161). In cells where
reprogramming is effective, silencing of INK4/ARF gene
transcription is observed concomitant with the induction
of molecular markers indicative of stem cell phenotypes (61).
Therefore, it is not surprising that iPS production efficiency
is increased by cellular immortalization (162), or short
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hairpin RNA (shRNA) knockdown of INK4/ARF transcripts
(61, 161). Currently, efficient generation of iPS from older
patients represents a major hurdle for regenerative medicine.
Therefore, novel reprogramming approaches aimed at curbing the senescent phenotype may improve iPS technology for
the future (163).
Genome-Wide Association Studies linking p16INK4a to
age-related disease
Meta-analysis of genome-wide association studies
(GWAS) suggests that we have only begun to scratch the
surface in understanding the role of p16INK4a in age-related
disease. Single-nucleotide polymorphisms (SNP) in chromosome 9p21.3 have been linked to cancer, atherosclerosis,
diabetes, frailty, cataracts. and late-onset Alzheimer’s disease
(Fig. 1B; ref. 164). In many cases, the expression of p16INK4a
correlates directly with SNP genotype, suggesting a causal
role for p16INK4a in diverse, age-associated diseases (164).
Several mechanistic models have been suggested to explain
how SNPs, some of which are >100 kb from the gene
promoter, influence p16INK4a expression. One model proposes that the activity of distal enhancer elements is modified
by SNP genotype (165). Another provides evidence that
ANRIL expression and splicing is directly influenced by
9p21 SNPs (166). We believe that these models are not
mutually exclusive.
Although mechanisms by which 9p21.3 SNPs influence
p16NK4a transcription have been proposed, the link
between p16INK4a expression and age-related diseases is
not always apparent. For example, atherosclerosis is frequently associated with lipid metabolism; yet, 9p21.3
SNPs have emerged as robust indicators of atherosclerotic
risk in some of the most widely replicated GWASs conducted to date (167). Evidence from animal models suggests that 9p21.3 SNPs may reduce INK4/ARF gene transcription, leading to altered cellular proliferation and apoptosis, which exacerbate disease progression (168–170).
However, transgenic mice carrying multiple copies of the
INK4/ARF locus are equally susceptible to atherosclerosis
(171). Therefore, the mechanisms by which 9p21.3 SNPs
influence a large number of age-related human disease
remain a subject of ongoing investigation.
Conclusions
Since the discovery of p16INK4a >20 years ago, numerous
advances have led to an increasingly complex view of
p16INK4a regulation and function. The role of p16INK4a
clearly extends beyond cancer and aging. Dynamic induction of p16INK4a is observed during mammary involution,
wound healing, nerve regeneration, and infection (unpublished data and refs 130, 172–174). It has been proposed that
the induction of p16INK4a during these highly proliferative
events is critical to the maintenance of proper tissue homeostasis. Whether the same signals that trigger p16INK4a
expression under physiologic conditions play a role in
tumorigenesis or aging is still unclear. However, p16INK4a
expression is only transient during processes like mammary
involution and wound healing (130, 172, 173). Are
p16INK4a-positive cells cleared by the immune system or
can they revert to a phenotype conducive to proliferation?
Understanding the role of p16INK4a in normal physiology
will be critical to the development of "senolytic" therapies,
which aim to lengthen our healthspan by eliminating senescent cells in the body.
p16INK4a is different from other INK4/ARF family
members. The dynamics of p16INK4a expression during
senescence make it a robust biomarker of mammalian
aging. Human tumors silence p16INK4a with greater frequency than ARF or p15INK4b (140), suggesting that the
tumor suppressor function of p16INK4a is somehow more
critical than that of the other INK4/ARF family members.
As such, the therapeutic restoration of p16INK4a activity
appears to be a promising avenue for antineoplastic
development. Ironically, although drug development
teams in the field of oncology work fervidly to move
CKD4/6 inhibitors into the clinic, aging biologists aim to
block the accumulation of p16INK4a-positive cells. Oddly,
the key to longevity likely lies in the hands of both groups,
as a careful balance of p16INK4a expression is required to
stave off cancer and prevent aging.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank M. Waqas, J. Gillahan, S.T. Nguyen, and Drs. D. Beach
(Bartholomews Hospital, United Kingdom), C.J. Burd (Ohio State University), and
N. Sharpless (University of North Carolina at Chapel Hill) for critical reading of the
manuscript. Dr. K. Hoadley (University of North Carolina at Chapel Hill) provided
advice and guidance regarding the analysis of TCGA data.
Grant Support
This work was supported by NIH R00AG036817 (C.E. Burd) and American
Cancer Society IRG-67-003-50 (C.E. Burd).
Received July 1, 2013; revised September 23, 2013; accepted September 24, 2013;
published OnlineFirst October 17, 2013.
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The Molecular Balancing Act of p16INK4a in Cancer and Aging
Kyle M. LaPak and Christin E. Burd
Mol Cancer Res 2014;12:167-183. Published OnlineFirst October 17, 2013.
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