New Insights into P53 Signalling and Cancer:
Implications for Cancer Therapy
Lo Nigro
Lo Nigro Cristiana, Laboratory Cancer Genetics and Translational Oncology, Department of
Oncology, S. Croce General Hospital, Via Carle 25, 12100, Cuneo, Italy
Correspondence
to: Lo Nigro Cristiana, Laboratory Cancer Genetics and Translational Oncology,
Department of Oncology, S. Croce General Hospital, Via Carle 25, 12100, Cuneo,
Italy.
Email:
lonigro.c@ospedale.cuneo.it
Telephone: +39-0171-616338
Fax: +39-0171-616331
Received: November 4, 2013
Revised:
December 4, 2013
Accepted: December 10, 2013
Published
online: January 18, 2014
ABSTRACT
The tumor suppressor p53 is one of the most highly studied proteins in the
field of cancer research because of its role in tumor cell survival and
apoptosis. Research over the past three decades has identified p53 as a
multifunctional transcription factor, which regulates the expression of
>2,500 target genes. p53 impacts myriad, highly diverse cellular processes,
including regulation of the cell cycle, maintenance of genomic stability and
fidelity, apoptosis, senescence and longevity, metabolism, angiogenesis,
cellular differentiation, and the immune response[1].
It is one of the
most important and extensively studied tumor suppressors. Approximately half of
human cancers have inactivating mutations in the p53 gene (known as TP53 in
human) and most of the remaining malignancies deactivate the p53 pathway by
increasing its inhibitors, reducing its activators or inactivating its
downstream targets. Activated by various stresses, including genotoxic damage,
hypoxia, heat shock and oncogenic assault, p53 blocks cancer progression by
inducing transient or permanent growth arrest, by enabling DNA repair or by
activating cellular death programs[2].
In addition to the
indisputable importance of p53 as a tumor suppressor, an increasing and
sometimes bewildering number of new roles for p53 have recently been reported,
including the ability to regulate metabolism, fecundity, and various aspects of
differentiation and development[3].
It is impossible
to cover all aspects of p53-associated biology in one review and so we have
reluctantly passed over many fascinating topics and we will focus on current
strategies and challenges to restore p53 tumor suppressor function in
established tumors and the therapeutic approaches designed to promote ore
deliver wild-type p53 function to cancer cells (i.e. adenoviral gene transfer
and small molecule activator of p53, to inactivate p53 inhibitors and to
restore wild-type function to mutant p53).
© 2014 ACT. All rights reserved.
Key words: p53; Complex biology; Signalling
network; Cancer therapy
Cristiana LN. New
Insights into P53 Signalling and Cancer: Implications for Cancer Therapy. Journal
of Tumor 2014; 2(1): 73-82 Available from: URL:
http://www.ghrnet.org/index.php/JT/article/view/590
INTRODUCTION
The tumour
suppressor TP53 (MIM# 191170), located on chromosome 17p13.1 is known as ¡®the
guardian of the genome¡¯ or ¡®the cellular gatekeeper of growth and division¡¯.
The gene contains 11 exons and transcribes a 2.8 kb mRNA, which is translated
into a 53 kDa phosphoprotein containing 393 amino acids. p53 is a key regulator
of cellular growth control and plays a central role in the induction of genes
that are important in cell cycle arrest and apoptosis following DNA damage[4].
Whilst the tumor suppressor
functions of p53 have long been recognized, so that p53 is one of the most
highly studied tumor suppressor genes in the field of cancer research, the
contribution of p53 to numerous other aspects of disease and normal biology and
physiology is only now being appreciated. This ever increasing range of
responses to p53 is reflected by an increasing variety of mechanisms through
which p53 can function, although the ability to activate transcription remains
key to p53¡¯s modus operandi.
p53 is a central hub in a
molecular network controlling cell proliferation and death in response to
potentially oncogenic stress conditions. A wide array of covalent
post-translational modifications and protein interactions regulate its
stability and sub-cellular localisation and modulate the nuclear and
cytoplasmic activities of p53[3].
The p53 relatives p73 and
p63 are entangled in the same regulatory network, being subject at least in
part to the same modifications and interactions that convey signals on p53.
Other p53 interactors exert an effect further downstream to directly modulate
p53 biological effects, for example, at the mitochondria[5].
One recent review lists 129
such p53 transcriptional targets that were identified as a result of either
single gene discoveries or multigene screens[6]. There are likely to
be many more genes specifically bound and activated by wild-type p53 (wtp53).
Furthermore, the number of genes whose expression is altered indirectly upon
induction of p53 is likely to be in the thousands, both with canonical[6]
and non-canonical[7] p53 response elements (REs).
This complex set of
molecular events actively contribute to the resulting cellular output and lead
to growth restraining responses. Control of p53¡¯s transcriptional activity is
crucial for determining which p53 response is activated, a decision we must
understand if we are to exploit efficiently the next generation of drugs that
selectively activate or inhibit p53[4].
With the molecular
elucidation of p53 signalling continuing to unravel novel concepts and broaden
our horizon of p53 function and the importance of p53 signalling for the
pathogenesis of cancer, drug development programs have begun to target the p53
signalling pathway.
In this review, we describe
the multi-faceted spectrum of p53 activities, discuss current strategies to
active p53 in tumors and will conclude with an outlook on future strategies and
challenges to translate p53-targeting therapies into clinical practice.
The complex biology of p53
Three decades of p53
research have produced more than 50,000 publications, which characterized p53
as a transcription factor orchestrating transcriptomic changes in response to a
broad spectrum of cellular stresses. In more recent years, it has become clear
that p53 function extends beyond canonical cell cycle, senescence and cell
death signalling[2].
Gene expression microarrays
have revealed that p53 associated-gene clusters impact additional, highly
diverse biological processes such as metabolism, aging, energy metabolism,
angiogenesis, immune response, cell differentiation, motility and migration and
cell¨Ccell communication[2]. Recent studies have demonstrated how
p53-dependent activation of microRNA genes can participate in the modulation of
various biological activities[7].
The complex repertoire of
p53 regulated genes further highlights the need to understand how p53 is
regulated and how selects its targets.
Regulate the regulator
p53 is regulated by
an array of posttranslational modifications both during normal homeostasis and
in stress-induced responses. More than 36 different amino acids within p53 have
been shown to be modified in various biochemical and cell culture studies[8].
Since the first discoveries
showing that p53 undergoes stress induced phosphorylation or acetylation, there
have been numerous complicated studies describing the modifications to p53 and
deciphering how they affect p53 function as a transcriptional regulator.
Phosphorylation of p53 is classically regarded as the first crucial step of p53
stabilization, but the tight control of cellular p53 levels is primarily
achieved through its ubiquitin-mediated proteasomal degradation. A number of
excellent reviews cover these aspects of p53 regulation[8-11].
p53 mutations and human cancers
As a tumour
suppressor, the major p53 functions are to regulate growth arrest and apoptosis
(see the review by Vousden and Prives[3]) (Figure 1) and the balance
of these two cellular events can determine the fate of individual cells. Unlike
other tumour suppressor genes, most TP53 mutations in tumours are of the
missense type and lead to single amino acid changes that predominantly affect
residues in the DNA binding domain of the protein, strongly suggesting that
targeted sequence-specific DNA binding is crucial for the escape of tumours
from p53 suppressor activity[7].
More than 26,000 somatic
mutation in TP53 appear in the international agency for research on cancer
(IARC) TP53 database version R14 (http://www-p53.iarc.fr/; the International
Agency for Cancer Research TP53 Mutation Database; TP53 Website).
The frequency of TP53
mutation varies from around 10% (hematopoietic malignancies) to 50-70%
(ovarian, colorectal, and head and neck malignancies)[4,12].
Germline mutations of TP53 cause Li-Fraumeni syndrome, which is a familial
cancer syndrome including breast cancer, soft tissue sarcoma, and various other
types of cancer[13]. Most TP53 mutations in human cancers result in
mutations within the DNA binding domain, thus preventing p53 from transcribing
its target genes. However, mutant p53 (mutp53) not only loses normal function
of the wild-type protein but also gains new abilities to promote cancer[14].
Somatic mutations at
individual residues have been associated with specific clinical phenotypes in
different type of cancer[15]. In addition, the spectrum of p53
deletions or mutations observed among tumor cells suggests that the mutations
vary in their prognostic power. Disruptive p53 mutations in tumor DNA are
reported to be associated with reduced survival following surgical treatment of
Head and Neck Squamous Cell Carcinoma (HNSCC)[16].
In addition to the presence
of somatic mutations, polymorphic features of the gene may also contribute to
alteration of normal p53 function and variants, mainly in the form of single
nucleotide polymorphisms, can be expected to impact tumor susceptibility to cytotoxic
drugs, radiation and chemo-radiation[4,17,18]. Elucidation of the
effect of TP53 polymorphisms is a challenge, which is attracting an
interest in the recent years. In fact, no uniform conclusion can be drawn for
roles of polymorphisms and mutations in the TP53 gene as results are so far
inconsistent.
p53 as a Sensor of DNA Damage:
apoptosis or cell cycle arrest and senescence
Environmental
hazards (e.g., UV sunlight, chemical mutagens, and oncogenic pathogens), and
cell-intrinsic metabolic processes can damage DNA. Such damage can alter DNA
structure and consequently gene transcription, or can cause mutations that
impact function. If left unrepaired, DNA damage can cause neoplastic growth.
There are very sophisticated systems for detecting DNA damage and repairing the
genome. p53 is normally in ¡®standby¡¯ mode and plays an important role in such
¡°caretaker¡± systems. This is why p53 is the so called ¡°guardian of the genome¡±[16].
p53 acts as an internal sentinel for DNA damage, and other cellular stresses,
including hypoxia, oncogene activation, starvation, altered mitochondrial and
ribosomal biogenesis, spindle poisons, or denuded telomeres. Depending on the
level of cellular compromise, p53 can either promote the repair and survival of
damage cells or promote the permanent removal of irreparable damage cells
through apoptosis or autophagy[19].
Many of our models for p53
function suppose that induction of programmed cell death is the key mechanism
by which p53 eliminates cancer cells. During cell cycle arrest, p53-regulated
pathways, including those involving growth arrest and DNA damage inducible 45
(Gadd45) and the p53 ribonucleotide reductase small subunit 2 (p53R2), are
significant in the repair of damaged DNA[16]. In the absence of
competent repair activity, DNA damage induces apoptosis (a) by transcriptional
activation of critical apoptosis regulators of the extrinsic, i.e. death
receptor-dependent, and the intrinsic, i.e. mitochondria-mediated apoptosis
signalling pathway, and (b) by directly impacting mitochondrial membrane
physiology via the intricate interplay with mitochondrial membranes and Bcl-2
family proteins[2].
p53-controlled apoptosis
involves transcriptional induction of components of the death receptor and
mitochondrial pathways including CD95, Puma, Noxa, Bax and others, which
cooperatively promote cell death. In addition, p53 protein can directly promote
mitochondrial outer membrane permeabilization (MOMP) to trigger apoptosis by
modulating the MOMP governing Bcl-2 family[20].
Early studies showed that
wtp53, functioning mainly as a transcription factor, can bind the Bax gene
promoter region and regulate Bax gene transcription. Bax is a member of the
Bcl-2 family, which forms heterodimers with Bcl-2, inhibiting its activity. The
Bcl-2 protein family plays an important role in apoptosis and cancer[21].
For example, Bcl-2 controls the release of cytochrome c from the mitochondria,
which activates the apoptotic pathway by activating caspase 9. Caspase 9 then
activates executioner caspase 3. Both caspases play key roles in the apoptotic
pathway.
Moreover, upon stress, a
p53-protein based mitochondrial apoptosis program may be activated and a
cytoplasmic pool of p53 rapidly translocates to the mitochondrial surface,
where it physically interacts with both anti- andpro-apoptotic Bcl-2 family
members to inhibit or activate their respective functions, leading to MOMP and
apoptosis. In this role, p53 acts like a BH3-only protein, either as direct
activator of the Bax/Bak effectors, or as sensitizer/de-repressor of Bcl-xL/2
and Mcl1[20].
More in details, PUMA
(p53-upregulated modulator of apoptosis) is a key mediator of the apoptotic
pathway induced by p53. When PUMA is disrupted in colon cancer cells,
p53-induced apoptosis is prevented. Thus, PUMA may play a pivotal role in
determining cell fate (programmed cell death versus cell cycle arrest) in
response to p53 activation[22]. CD95 (also called Fas and Apo-1) is
a ¡°death receptor¡± indicating its major role in apoptosis. Several reports have
indicated the CD95 pathway to play an important role in apoptosis induced by
cytotoxic agents, and that this system involves the activation of wtp53[23].
Therefore, the p53 status may influence chemosensitivity via CD95 signalling.
However, a recent report indicated that CD95 could promote tumor growth[24].
By contrast, the
p53-regulated Ring domain E3 ubiquitin ligase MDM2 (murine double minute 2)
functions to produce negative feedback, which regulates p53 activity[25].
MDM2 recognizes a short region in the TA domain of p53 and interferes with its
transcriptional activity; at the same time, MDM2 interacts with the DBD region
and ubiquitinates p53, promoting its proteasomal degradation. As MDM2 is a
transcriptional target of p53, inhibition by MDM2 is part of a negative
feedback loop on p53 activation[26].
In addition, depending on
the type of cellular stress, p53 can induce G1 arrest through activation of
transcription of the cyclin-dependent kinase inhibitor p21. The p21/WAF1 (wtp53
activated fragment 1) gene product, a p53 target gene, inactivates the
proliferating cell nuclear antigen (PCNA), which can regulate DNA replication,
and induce a p53-dependent G1 arrest through the inhibition of cyclin/CDK
activity[16].
In the presence of cellular
stresses, p53 is subjected to a complex and diverse array of covalent
post-translational modifications. These include phosphorylation, acetylation,
poly (ADP-ribosyl) ation, ubiquitylation and sumoylation. In response to
cellular stress, Ser15/Ser20 in p53 are phosphorylated and MDM2 is separated
from the phosphorylated p53, leading to the stabilization and activation of
p53. Therefore, p53 can bind to the promoter of the p21 or p53R2 genes
associated with DNA repair, and induce their expression[16].
It is currently unknown
whether p53 can also activate oxidative stress-induced necrosis. Intriguingly,
recent findings provide genetic, biochemical and pharmacological evidence that
fundamentally expands our understanding of p53-mediated cell death networks
into necrosis An unexpected critical role of stress-accumulated mitochondrial
p53 protein in directly regulating permeability transition pore (PTP) at the
inner membrane has been described.
Upon oxidative stress, p53 triggers PTP opening by engaging in a
physical interaction with Cyclophilin D (CypD), thereby inducing necrotic cell
death in mouse and human cells[20].
However, transient cell
cycle may not lead to tumor eradication, because a cell with oncogenic
potential that cannot be repaired may resume proliferation[27].
Therefore, the other mechanism, cellular senescence, may play an important role
in p53-mediated tumor suppression. Cellular senescence is permanent cell cycle
arrest. There are many reports regarding the correlation between tumor
development, p53 and senescence[28]. The inactivation of p53, as is
present in most human cancers, allows cells to evade cellar senescence, thus
resulting in tumor development. How precisely p53 determines whether or not the
activation of the senescence program or the apoptosis program occurs still
remains to be elucidated. This question is especially important for the
development of p53-based cancer therapy, including approaches in combination
with conventional chemotherapy. Most conventional chemotherapeutic agents
achieve elimination of cancer cells by killing them. Therefore, if p53 induces
senescence rather than apoptosis, a conflict will emerge.
Vousden and Prives[3]
proposed a model wherein the decision between life and death can be determined
by the extent of damage or the duration of stress. In their model, a low level
of stress which can be repaired elicits a DNA repair/survival response, while a
high level of stress that cannot be repaired induces an apoptotic or senescence
response. This dual nature of p53, killer and protector, indicates the
possibility that p53 may also act as tumor promoter. The anti-apoptotic
function of p53 may lead to the survival of damaged cells, which may increase
the possibility for malignant transformation (Figure 2).
p53 and cell survival
In addition to
eliminating damaged cells, p53 can also contribute to cell survival through a
surprisingly large number of mechanisms[3]. Numerous p53 target
proteins function to inhibit apoptosis, including p21, decoy death receptors
such as DcR1 and DcR2, the transcription factor SLUG (which represses the
expression of PUMA), and several activators of the AKT/PKB (protein kinase B)
survival pathways[29]. Another group of p53-inducible genes have
recently also been shown to act as antioxidants by decreasing the levels of
intracellular reactive oxygen species[30,31]. Although this function
for p53 would help inhibit tumor progression by protecting cells against DNA
damage and genome instability, down-regulation of reactive oxygen species
through these p53-dependent mechanisms can also result in decreased
susceptibility to apoptosis[32].
p53 and autophagy
An emerging
non-nuclear function of p53 is in regulation of autophagy, a process that
allows removal of damaged cytoplasmic organelles and adaptation of cells to
metabolic stress. Although p53 can transactivate genes that induce autophagy
under stress conditions (e.g, DRAM, TSC2, Sestrin1 and 2, PTEN, and IGFBP3),
depletion or mutation of p53 actually increases autophagy, suggesting that p53
constitutively limits this process in normal growing cells[33,34].
Even if the mechanism remains unknown, the autophagy-inhibitory activity is
ascribable to the cytoplasmic pool of p53, as degradation of cytosolic p53 by
MDM2 promotes autophagy after nutrient depletion, endoplasmic reticulum stress,
or treatment with rapamycin[5,35]. In fact, p53 can be activated by
metabolic adversity (such as starvation) - a response that can be mediated
through the action of AMP-activated protein kinase (AMPK), a key component of
the cell¡¯s response to bioenergetic stress[36]. p53 then promotes a
program of gene expression (including the induction of AMPK expression) to
negatively regulate the kinase mTOR (mammalian target of rapamycin), a central
node in the control of protein synthesis[3,33,37].
p53 and the regulation of ribosome
biogenesis
In a growing cell,
ribosome biogenesis is a major consumer of cellular energy and resource. Thus,
as growth conditions change, cells must rapidly rebalance ribosome production
with the availability of resources. It has been shown that serum starvation
activates p53 and induces cell cycle arrest in an RPL11 (L11)-dependent manner
through a mechanism involving translocation of L11 of from the nucleolus to the
nucleoplasm[38]. The model derived from a myriad of in vitro
data suggested that when cells sense nutrient-shortage stress, ribosomal
proteins are released from the nucleolus to the nucleoplasm where they can bind
to MDM2 and inhibit its E3 ligase activity, leading to activation of p53[39].
The relevance of these findings was further substantiated by the discovery of
MDM2 mutations in the region that binds to L11 in human cancers. As a result,
the MDM2 mutants are refractory to inhibition by ribosomal proteins and are
maintained in a p53-suppressive mode[40].
p53, tumor glycolysis and fatty acid
metabolism
Recent observations
show that many tumor suppressor genes play important roles in metabolic
regulation, in addition to their established roles in cell survival and
apoptosis[19]. Cancer cells are characterized by aerobic glycolysis
with the use of glucose and production of lactate. Several biologic functions
of p53 decrease the glycolysis pathway in cells. p53 induces TP53-induced
glycolysis and apoptosis regulator (TIGAR) expression via transcriptional
activation. Moreover, wtp53 downregulates phosphoglycerate mutase (PGM), and
mutation of p53 can enhance PGM activity and glycolytic flux[41,42].
In addition, p53 can reduce
intracellular glucose levels by inhibiting the expression of glucose
transporters. For example, p53 directly represses the transcriptional activity
of the GLUT1, GLUT3 and GLUT4 gene promoters[43].
p53, aging and stem cells
Cancer is an
age-related disease. Consequently, genes that stop cancer progression promote
longevity by restraining genomic instability, and inhibiting the growth and
expansion of genetically aberrant cells. p53 has a prominent - and
controversial - role in the regulation of ageing and longevity. Excessive p53
tumor suppressive activity, however, can be detrimental to organism homeostasis
by promoting certain aspects of aging[2]. Maintenance and
regeneration of adult tissues, and consequently longevity depend on the
continuous proliferation and differentiation of resident stem cells[44,45].
In line with a tumor suppressive, pro-death and anti-proliferative role of p53,
deficiency in p53 was shown to attenuate profound defects in tissue homeostasis
caused by mutations in DNA repair genes. Some studies suggest that moderate p53
activation is beneficial for tissue homeostasis, as chronic hyperactivation of
p53 decreases longevity, while moderate, physiological enhancement of p53
activity with intact regulatory mechanisms to control p53 stability, induces an
anti-aging phenotype. It appears that duration and extent of stress and
consequently levels p53 activity determine cell and organism fate; high p53
activation restricts proliferation, low levels induce cell survival, and
decrease oxidative damage via induction of an antioxidant gene signature[46,47].
p53 and miRNA
Protein-encoding
genes are not the only transcription targets of p53. Several groups
independently reported that p53 can directly regulate the expression of
specific microRNAs (miRNAs), most dramatically the miR-34 locus consisting of
miR-34a, miR-34b, and miR-34c[48-51]. Several reports showed that
p53 can bind directly to response elements within the miR-34a and miR-34b/c
promoters to stimulate transcription from this locus. Certainly, miR-34a
expression is physiologically relevant to the impact of p53 activity on cells:
it can induce cell cycle arrest and senescence, as well as facilitate cell
death[3] .
It has now been reported
that miR-192 and miR-215 are also induced by p53 and promote increased p21
expression[49]. Moreover, miR-145 has been implicated as a p53
target that can repress c-myc expression[52].
CURRENT STRATEGIES TO TARGET THE P53 PATHWAY
In tumor cells, the
p53 pathway is often disrupted. Therefore, recovering the function of wtp53 and
its targets in tumor cells is a key therapeutic objective; the obvious goal is
to try to restablish the growth-inhibitory functions of p53 in cancer cells.
Indeed, reintroduction of
wtp53 using a replication-defective adenoviral vector showed efficacy in
reversing the growth of several human tumor types, demonstrating that
restoration of p53 activity is a viable anticancer therapeutic approach.
However, as regulation of p53 activity becomes better understood, approaches
that exploit our deeper understanding of the biochemistry of p53 activation
have led to the identification of small molecules that can manipulate the
endogenous non-functional protein that is so often expressed in tumor cells. As
a result, strategies have focused on restoring wild-type activity to the mutp53
protein, restoring functionality of the p53 pathway or activating one of the
p53 family members.
In this review, we have
described the multi-faceted spectrum of p53 activities; now we will discuss
current strategies to activate p53 in tumors, with an outlook on future
strategies and challenges to introduce p53-targeting therapies into clinical
practice.
In particular, cancer
therapies aimed at targeting signalling pathways controlled by p53 include
p53-gene therapy, chemical chaperones, p53 C-terminal peptides and small
molecules that can target p53. Some therapeutic strategies are independent of
p53 status in cancer cells, including high-linear energy transfer (LET)
heavy-ion radiation; others involve enhancement of cancer therapies with different
strategies, including an RNA-silencing therapy targeted at DNA repair pathways
and a molecular-targeting therapy for the survival pathway Akt-mTOR (Ota 2012)
(Figure 3).
p53 gene therapy
In recent years, one
line of attack that has been successful in the clinic is the introduction of
exogenous wtp53 into cancer cells, either by gene delivery or by direct protein
delivery[16,53] has been explored.
The first p53-based gene
therapy was reported in 1996. A retroviral vector containing the wild-type p53
gene under the control of an actin promoter was injected directly into tumors
of non small cell lung cancer patients[54]. After development of a
replication-defective recombinant p53 virus (Ad5CMV-p53)[3], many
clinical subsequent trials have been performed.
Although preliminary studies
in cell cultures and in animal models have indicated the potential viability
and low toxicity of these approaches[55-58], their efficacy in
clinical trials is currently controversial. Clinical studies in lung, bladder,
ovarian and breast cancer showed no beneficial effects compared to conventional
treatments. On the other hand, encouraging results were reported in HNSCC,
where p53 mutations are frequent, and their incidence increases with
progression (for review: Ota et al, 2012[16]; Shen et al,
2012[19]).
Because of its affinity for
the cells of the upper aerodigestive tract, a modified adenovirus has been the
most widely-used vector for p53 gene therapy in HNSCC (AdCMV5-p53; INGN 201)[59,60].
Therefore, a recombinant human adenovirus that expresses functional wtp53 has
been approved by the Chinese government for the treatment of HNSCC[25,61].
Treatments showed that antitumor efficacy was associated with the expression
and activity of functional p53, and adverse effects were also significant[54,62-64].
For a complete review on the current p53-based therapeutics for HNSCC, refer to
Tassone et al[65] (Figure 4).
Although such results are
encouraging, further improvements in methods are required to accomplish the
safe and effective delivery of wtp53 in vivo[16,66].
Another way to eliminate
cells with mtp53 is to deliver a virus that preferentially target cells that
lack functional p53. Adenoviruses contain an E1B gene producing a 55 kD protein
that inactivates host p53 to promote host cell survival. Extensive in vitro
and in vivo studies showed that Onyx-015, an adenovirus lacking the E1B
gene, developed to selectively eliminate cells without functional p53, is
capable of replicating in and promoting the lysis of carcinoma cells. In the
absence of wtp53 activity in cancer cells, the generation of a mutated viral
vector for tumor cell lysis (as Onyx-015) was exploited[16,64,65,67].
Accordingly, the Onyx 015 reagent, a p53-targeting oncolytic mutant adenovirus,
has been developed for clinical application. However, evaluation of numerous
clinical trials performed thus far have indicated that the administration of
Onyx-015 as a single agent produces only marginal benefit, whereas its
administration in combination with conventional therapy is more effective[68].
Small molecules and chaperones
p53 Stabilization: Our growing understanding of how p53 is
regulated has also led to the development of small molecule drugs that
stabilize and activate the p53 protein.
As can be envisaged, the
MDM2-p53 interplay is a particularly attractive target for therapeutic
intervention in cancer. Increasing the expression and activity of wtp53 is the
ultimate goal in most treatment strategies. MDM2 is an E3 ubiquitin ligase
which controls p53 degradation via ubiquitylation[19,69,70]. Many
tumors overexpress MDM2, even tumors without p53 mutations[71]. In
fact, due to the importance of the MMD2-p53 interaction, inhibition of this
event with small molecules is regarded as having therapeutic potential. A
number of different strategies have been employed to screen for and develop
small molecules that bind specifically to the N-terminal region of MDM2 that
interacts with p53[3,25,65,72].
In particular, MDM2
inhibitors HLI98 and Nutlin 3A can, respectively, stabilize p53 and rescue
tumor suppression function in solid tumors[19,73-75] and in
hematological malignancies[76].
The nutlins are
cis-imidazoline compounds that act as antagonists of the MDM2-p53 interaction.
Analysis of the crystal structure showed that nutlin binds in the pocket of
MDM2 to prevent the p53-MDM2 interaction. Nutlin can activate the p53 pathway,
thereby inducing cancer cells and xenograft tumors in mice to undergo cell
cycle arrest, apoptosis and growth inhibition[71,77]. For a review
highlighting recent advances in the development of small-molecule MDM2
antagonists as potential cancer therapeutics, with special emphasis on
Nutlin-3, refer to Shen et al[78].
MI-219 binds to the p53
binding pocket in MDM2 and disrupts the MDM2¨Cp53 complex, which leads to
activation of p53, induction of growth arrest and apoptosis and suppression of
xenograft tumor growth[75]. MI-219 also activates the p53 pathway in
cells with wtp53. Apoptosis and cell cycle arrest were observed in xenograft
tumors which resulted in tumor regression[71,79]. Unfortunately,
this approach carries the risk of enhancing the pro-survival adaptation
functions of p53 in some tumors[80,81]. Clarifying the mechanism(s)
by which p53 coordinates adaptation could lead to the discovery of new
therapeutic targets in cancer expressing wtp53.
Tenovin was found by a
cell-based drug screen to activate p53. Tenovin acts as an inhibitor of the
NAD+-dependent class III histone deacetylating activities of SirT1 and SirT2,
two important members of the sirtuin family. Intra-peritoneal administration of
tenovin-6 has been demonstrated to induce a regression of xenograft tumors in a
mouse mode[82].
Issaeva et al (2004)
screened a chemical library and found the small molecule RITA (reactivation of
p53 and induction of tumor cell apoptosis), which binds to p53 and inhibits the
p53-MDM2 interaction both in vitro and in vivo. RITA is reported
to inhibit glycolytic enzymes and, therefore, induce robust apoptosis in various
cancer cells, including leukemic cells, that retained wtp53[55,65,83,84].
They also found that the p53 released from MDM2 by RITA promotes p21 and hnRNP
K (a p53 cofactor), thus implying that p21 plays a major role in regulating
cancer cell fate after p53 reactivation[85].
p53 Restoration: Another approach in preclinical
development involves restoring tumor-suppressing function to mp53. Studies have
demonstrated that glycerol, as a chemical chaperone, can restore normal p53
function in mtp53 HNSCC[16].
There is class of small molecules
that reactivate the wild-type functions of mtp53. PhiKan083 is a carbazole
derivative found from in silico screening of the crystal structure of p53. By
binding mtp53, PhiKan083 raises the melting temperature of mtp53, which results
in the reactivation of its function[86].
PRIMA-1 (or ¡®¡®p53
reactivation and induction of massive apoptosis¡±) is another small molecule
identified by cell-based screening which restored sequence-specific DNA binding
and the active conformation of p53. It is known to induce apoptosis through the
p53-dependent c-Jun-NH2-kinase pathway[85,87].
CP-31398 is also a small
qinazoline based molecule that was found to produce an active p53 in cancer
cells and can restore the protein folding of mtp53 to a more natural
conformation that permits a wild-type function[16,88]. CP-31398
stabilizes the DNA-binding domain of p53 for both wt and mt (V173A and R249S)
p53[89].
Molecules that disrupt exogenous p53
inhibitors: The causative role
of human papillomavirus-16 (HPV16) in HNSCC is largely attributed to two HPV16 oncogenes, E6 and E7. Since
inactivation of p53 by HPV16 E6 is critical for HPV-mediated tumorigenesis,
reactivation of p53 may be an efficient strategy to eliminate HPV16-positive
HNSCC cells. Recent work has identified CH1iB as a small molecule that disrupts
the interaction between HPV16 E6 and p300 in HPV 16-positive UMSCC47 and
UPCI-SCC090 HNSCC cells[65,90]. CH1iB increased total and acetylated
p53 levels, enhanced p53 transcriptional activity, and increased the expression
of p53-regulated genes, p21, miR-34a, and miR-200c.
Alternative targets
The discovery that
the p53 family members p63 and p73 have similar structures and have similar
biological activities has provided an additional anti-tumor strategy. Both p63
and p73 can induce apoptosis and do so by activation of some pro-apoptotic
targets as wtp53[91]. Importantly, mutational inactivation of p63
and p73 is rare in human tumors and they are widely expressed, making these
proteins attractive chemotherapeutic targets. Indeed, results from recent
studies demonstrate that targeting these proteins may be a useful anticancer
approach[75].
Importantly, screens for
proteins that interact with p53 have identified a family of proteins, termed
ASPP, which can augment the ability of p53 to stimulate the expression of
proapoptotic genes. One of these family members, iASPP, suppresses the activity
of p53, p63 and p73 by interacting with their DNA binding domains[92].
Characterization of the activity of 37AA, a p53-derived peptide termed 37AA
which could drive cell death through activation of p73, demonstrated that it
functioned by interfering with iASPP binding with p73 and promoted its ability
to stimulate the expression of proapoptotic genes such as PUMA and NOXA[93].
p53-Based Immunotherapy
Other strategies to
restore wild-type p53 in the cell have been vaccines against mtp53, small
mol¬ecules that bind to mtp53 to restore normal conformation and/or activity
(e.g. ellipticine)[94]. p53 protein, especially mtp53, may be a
target of tumor antigen specific cytotoxic T lymphocytes that can mediate
immune response of host against cancer in vivo[95]. Some
cancer patients have antibodies against p53[96,97], although the
frequency and clinical significance are still under debate[16].
Speetjens et al[98]
reported clinical trials of a p53-specific synthetic long peptide (p53-SLP)
vaccine for metastatic colorectal cancer patients[99], where ten
patients were vaccinated with p53-SLP in a Phase I and Phase II trial.
Preclinical phase I/II trial of INGN-225 (Introgen), a p53-modified
adenovirus-induced dendritic cell vaccine for small cell lung cancer (SCLC)
patients, has been reported[100].
Targeting tumor metabolism
In the last years,
the emerging role of p53 in tumor metabolism has suggested that drugs that
mimic the metabolic effect of p53 are able to perturb cancer cell metabolism
and inhibit cancer cell proliferation[19]. Because tumor cells rely
on glycolysis or ATP production for their survival, the molecular targets of
p53 in the glycolytic pathway might be potential therapeutic targets in cancer.
Indeed, the non-metabolizable glucose analogues 2-deoxyglucose or
3-bromopyruvate can inhibit glycolysis and ATP production[101,102].
Moreover, the glucose transporter inhibitor phloretin inhibits glucose uptake
and sensitizes tumor cells to the chemotherapeutic drug daunorubicin[103].
As p53 repression of GLUT3 expression is mediated by the IKK¨CNF-B pathway, inhibition of the activation of the IKK¨CNF-B pathway can, thereby, be another target for cancer
treatment. R-roscovitine has been shown to inhibit the function of IKK and
downregulate NF-B activation. In addition to the NF-B pathway, the cyclin-dependent kinase inhibitor
roscovitine can also dramatically enhance the expression of p53 and block the
degradation of p53 mediated by MDM2, thereby activating the p53 pathway and
inhibiting glycolysis in tumors[104-106].
The activation of AMPK induces
fatty acid oxidation and mitochondrial respiration and represses fatty acid
synthesis and glycolysis. Thus, AMPK may be a beneficial target for cancer
treatment. Recent studies support this finding, showing that pharmacologic AMPK
activators, such as metformin, phenformin, and AICAR, attenuate cancer cell
growth and inhibit tumorigenesis in animal models[107,108].
CONCLUSION
Research over the
past three decades has identified p53 as a multifunctional transcription
factor, which regulates the expression of >2,500 target genes. p53 impacts
myriad, highly diverse cellular processes, including the maintenance of genomic
stability and fidelity, metabolism, longevity, and represents one of the most
important and extensively studied tumor suppressors. Activated by various
stresses, foremost genotoxic damage, hypoxia, heat shock and oncogenic assault,
p53 blocks cancer progression by provoking transient or permanent growth
arrest, by enabling DNA repair or by advancing cellular death programs. This
potent and versatile anti-cancer activity profile, together with genomic and
mutational analyses documenting inactivation of p53 in more than 50% of human
cancers, motivated drug development efforts to (re-) activate p53 in
established tumors. Thus it is indisputable that p53 represents an attractive
target for the development of anti-cancer therapies. Whether p53 is
¡®druggable¡¯, however, remains an area of active research and discussion, as p53
has pro-survival functions and chronic p53 activation accelerates aging, which
may compromise the long-term homeostasis of an organism. Thus, the complex
biology and dual functions of p53 in cancer prevention and age-related cellular
responses pose significant challenges on the development of p53-targeting
cancer therapies.
In this paper, we have focused on
the functions of p53 and therapeutic approaches targeting p53 for cancer
therapy. However, despite recent advances in the research on p53¡¯s function, it
appears that various questions still remain to be answered before the full
therapeutic of pharmacological modulation of p53 can be harnessed.
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Peer reviewers: Zhenghe John Wang,
Associate Professor, Department of Genetics and, Case Comprehensive Cancer
Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio
44106-7285, the United States; Chen Zhao, Susan G Komen Fellow, Lorry Lokey
Institute for Stem Cell Biology and Regenerative Medicine; G3115M , 265 Campus
Dr. Stanford, CA 94305, the United States; Chinmay Kumar Panda, Ph.D, Assistant
Director Grade, Head, Department of Oncogene Regulation, Chittaranjan National
Cancer Institute, 37 S. P. Mukherjee Road, Kolkata 700 026, India.
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