2502 HYPOTHESIS
Development 140, 2502-2512 (2013) doi:10.1242/dev.084210
© 2013. Published by The Company of Biologists Ltd
Tumor suppressors: enhancers or suppressors of
regeneration?
Summary
Tumor suppressors are so named because cancers occur in their
absence, but these genes also have important functions in
development, metabolism and tissue homeostasis. Here, we
discuss known and potential functions of tumor suppressor
genes during tissue regeneration, focusing on the evolutionarily
conserved tumor suppressors pRb1, p53, Pten and Hippo. We
propose that their activity is essential for tissue regeneration.
This is in contrast to suggestions that tumor suppression is a
trade-off for regenerative capacity. We also hypothesize that
certain aspects of tumor suppressor pathways inhibit
regenerative processes in mammals, and that transient targeted
modification of these pathways could be fruitfully exploited
to enhance processes that are important to regenerative
medicine.
Key words: Regeneration, Evolution, Tumor suppressors,
Differentiation, Tissue homeostasis
Introduction
Regeneration of complex tissues and organs after injury requires
extensive cellular proliferation that is precisely regulated and
executed in concert with differentiation and patterning mechanisms
that preserve the organization of tissues within an organ. How this
occurs with high fidelity, on both large and small scales, is matched
in sophistication and complexity only by organ generation during
development. Neoplasia represents a loss of control or absence of
these processes of proliferation or differentiation, respectively.
Tumor suppressor genes, initially identified because they are
inactivated in mammalian tumors, were subsequently shown
experimentally to protect against neoplastic transformation, and are
now known to have a long evolutionary history with the ability to
regulate diverse and fundamental cellular processes, including
growth and division, genome maintenance, differentiation,
metabolism and death (Fig. 1) (Sherr, 2004; Pearson and Sánchez
Alvarado, 2008). Many tumor suppressor genes play crucial roles
during normal development and postnatal life that are more likely
to explain why they evolved than does their role in protection from
tumors. The pervasiveness of tumor suppressor gene function
extends to include the context of tissue regeneration.
In this Hypothesis article, we discuss the mechanisms of action
and consequences of the diverse functions of tumor suppressors
1
Department of Surgery, Division of Plastic and Reconstructive Surgery, 2Department
of Orofacial Sciences, 3Craniofacial and Mesenchymal Biology Program, and 4Eli and
Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University
of California, San Francisco, CA 94143, USA. 5Baxter Laboratory for Stem Cell
Biology, 5Department of Microbiology and Immunology, 7Institute for Stem Cell
Biology and Regenerative Medicine, and 8Clinical Sciences Research Center, School
of Medicine, Stanford University, Stanford, CA 94305, USA.
*Authors for correspondence ([email protected];
[email protected])
during regenerative processes. In early vertebrates, the use of
proliferative progenitor and stem cells for tissue homeostasis and
regeneration may have also introduced a robust new source of
cancers, necessitating the development tumor suppression (Pearson
and Sánchez Alvarado, 2008; Belyi et al., 2010). It could be
surmised that this resulted in the creation of a perpetual ‘fine line’
on either side of which the need for proliferation must be balanced
against protection from cancer. Tumor suppressor mechanisms that
police stem cells and eliminate aspiring cancer cells might be
expected to impede proliferation and thus inhibit certain
regenerative processes, a form of antagonistic pleiotropy (Greaves,
2007). Therein lies a potential trade-off between tumor suppression
and regenerative capacity that is particularly highlighted when
examining the relationships between aging, cancer susceptibility
and stem cell proliferation (Kim and Sharpless, 2006; Campisi and
d’Adda di Fagagna, 2007; Seifert and Voss, 2013).
Tumor suppressor genes are subject to continuous evolutionary
pressure and their expression and activities are also modified in a
context-dependent manner. Thus, among vertebrates, tumor
suppressor pathways have variable components in different species
(del Arroyo and Peters, 2005; Belyi et al., 2010) and, within a
species, tumor suppressor gene function varies with developmental
stage and age (Kim and Sharpless, 2006). Regenerative capacity
also varies markedly among these same parameters of species,
developmental stage and age (Brockes and Kumar, 2008).
Although there is no readily identifiable hierarchical pattern of
regenerative capacity in vertebrate evolution, lower vertebrates that
are highly regenerative (such as teleost fish and urodele amphibians
– salamanders) and poorly regenerative vertebrates, such as
mammals, have differences in their tumor suppressor machinery
that could be related to their ability to regenerate.
We propose that, in a context-specific manner, some tumor
suppressor genes may exhibit ‘regeneration suppressor’ activities
that, if understood, could be targeted to improve regenerative
capacity. However, we also suggest that tumor suppressors are, in
general, necessary components for regeneration that both
orchestrate major aspects of the process and ensure its safety. The
current emphasis in the field of regenerative medicine on cellular
mechanisms of regeneration, including in vitro expansion and
reprogramming, has rekindled interest in the regulation of
proliferation, survival and cell death – processes that are the
domain of tumor suppressor genes. This also provides an
opportunity to revisit basic questions concerning how these genes
function in model organisms during regeneration in vivo.
As a comprehensive review of the many tumor suppressor genes
is not possible here, we focus specifically on four core tumor
suppressors, retinoblastoma (pRb1), p53 (Trp53), phosphatase and
tensin homolog (Pten) and Hippo (Box 1), for which substantial
published data exist, and which exemplify the diverse roles of tumor
suppressors during regeneration. We are primarily concerned with
regeneration in vertebrates, although we also touch on data from
Drosophila and planarians that illustrate relevant principles.
DEVELOPMENT
Jason H. Pomerantz1,2,3,4,* and Helen M. Blau5,6,7,8,*
HYPOTHESIS 2503
Development 140 (12)
Metabolism
Fig. 1. Known cellular functions of tumor
suppressors that could impact regeneration. The
Hippo, retinoblastoma, p53 and Pten pathways together
have diverse cellular functions. As discussed in the text,
some of these functions have been demonstrated to
impact on regenerative processes, whereas others have
yet to be studied in the context of regeneration. The
lightning bolts represent cellular stress, and different
cellular outcomes are shown depending on the degree
of stress to which the cell is exposed.
Migration and polarity
Growth and
protein synthesis
Tumor suppressors
Differentiation and maturation
ROS
ROS
Proliferation
Repair and survival
Susceptibility of regenerative vertebrates to
tumors
It has been suggested that the evolution of more complex tumor
suppressor pathways in higher vertebrates might be linked to size
and lifespan: that the greater number of cell divisions in a large,
long-lived animal would increase the chances of cancer. However,
such arguments are not tenable. For example, although humans are
large and have long lives, other mammals with similar tumor
susceptibility and regenerative capacities (or deficiencies) are small
and relatively short-lived. Moreover, comparison among highly
and poorly regenerative vertebrates does not indicate any strong
relationship between lifespan, size, regenerative capacity and
cancer resistance (discussed further below). Laboratory mice live
for 1-2 years in captivity, in contrast to zebrafish and axolotl, which
live from 2-4 years and 10-14 years, respectively. Although
zebrafish are small, axolotls are larger than mice. Therefore, when
considered objectively, a general basis for a greater need for tumor
suppression in higher vertebrates is not readily forthcoming. It is
possible that evolutionary pressure on tumor suppressor pathways
relates to environmental influences, differences in metabolism and
or immune systems. Nonetheless, it is difficult to identify a wellfounded teleological explanation for a trade-off relationship
between cancer susceptibility and regenerative capacity, at least
within vertebrates.
The notion that genetic divergence of tumor suppressor
pathways among highly regenerative (e.g. teleost fish and urodeles)
and poorly regenerative vertebrates (e.g. mammals) might be partly
responsible for differences in regenerative capacity begs the
question of whether regenerative vertebrates are susceptible to
tumors. The simple answer to this is yes. However, the issue is
more complex because the significant differences in environment,
physiology, anatomy, metabolism, etc., mean it is of limited
relevance to compare directly the incidence, penetrance and
phenotype of various tumors among fish, amphibians, rodents and
humans in a meaningful way. There is no question that a wide
variety of tumors occur in regenerative organisms both in the wild
and in captivity. Moreover, many of the tumor-promoting agents
used in mammalian studies are also tumorigenic in teleosts and
urodeles, and genetic models, especially in zebrafish, support the
existence of conserved genetic elements related to both tumor
formation and tumor suppression (Amatruda et al., 2002; Liu and
Leach, 2011). Virtually all tumor types described in humans have
been observed in zebrafish in captivity (Spitsbergen et al., 2012).
In addition, both anuran (frogs and toads) and urodele amphibians
develop neoplasms in all major organ systems (Anver, 1992). The
comprehensive chapter by Green and Harshbarger in Amphibian
Medicine and Captive Husbandry (Green and Harshbarger, 2001)
catalogs described amphibian tumors in the wild and in captivity,
bringing together multiple streams of data and applying stringent
criteria, including rigorous pathological analysis, to permit
confirmation of the diagnosis of ‘tumor’. Interestingly, in addition
to being documented in the viscera and hematopoietic systems,
spontaneously occurring benign and malignant tumors have been
found in urodele tissues and in cell types that are involved in
epimorphic regeneration, such as skin (papillomas and malignant
melanophoromas) and mesenchymal tissue (fibromas and
fibrosarcomas) (Wright and Whitaker, 2001). Taken together, the
available data do not indicate a clear generalized resistance or
enhanced susceptibility of urodeles or teleosts to tumor formation
or to cancer compared with other animals, and show that
regenerative vertebrates are susceptible to a broad spectrum of
malignancies.
However, some aspects of tumor susceptibility, particularly in
the contexts of exposure to chemical carcinogens and of the
specific tissues undergoing regeneration and blastema formation,
do suggest distinct responses at least in the urodele to factors that
would induce tumor formation in mammals or adult anuran
amphibians (reviewed by Tsonis, 1983; Anver, 1992; Brockes,
1998). In studies beginning in the 1940s in which chemical
carcinogens were applied to salamanders, sometimes directly to the
blastema (reviewed by Brockes, 1998), the surprising finding was
a lower than expected incidence of malignant tumor formation,
leading to the notion that urodeles have greater tumor resistance
than other species. Remarkably, transplantation of established
tumors into a regenerating blastema could even induce
differentiation and loss of the malignant phenotype. Generally, the
application of carcinogenic substances to regenerating structures in
urodeles interferes with regeneration and results in sprouting of
supernumerary limb buds, or other limb anomalies, but does not
cause invasive tumor formation (Breedis, 1952; Tsonis and Eguchi,
1981; Tsonis, 1983; Pfeiffer et al., 1985). In the regenerating newt
lens, only the regenerating dorsal iris is resistant to chemically
DEVELOPMENT
Apoptosis
2504 HYPOTHESIS
p53
The p53 protein is a sequence-specific transcriptional activator that
is normally expressed at low levels. p53 is stabilized and
accumulates in response to various forms of cellular stress (including
radiation, hypoxia, reactive oxygen species and oncogene
activation), triggering downstream gene expression. Depending on
the cellular context and degree of stress, activated p53 induces
either growth arrest and repair, autophagy, senescence or
apoptosis. The phenotypic end result is either cell recovery and
survival, or death. The principal negative regulator of p53 in
vertebrates is Mdm2, which can in turn be inhibited by the higher
vertebrate-specific tumor suppressor Arf. Thus, Arf activates p53,
causing induction of p21 (Cdkn1a) among other p53 target genes.
Retinoblastoma protein
Retinoblastoma protein (pRb1) is a ubiquitously expressed
transcriptional repressor. It blocks proliferation at the G1/S transition
by binding to and inactivating members of the E2F transcription
factor family. Phosphorylation and inhibition of pRb1 by cyclindependent kinases (CDKs) releases E2F and results in entry into S
phase. CDKs are in turn inhibited by the inhibitor of kinase (INK)
family of proteins, of which Ink4a is a principal member and
important tumor suppressor. pRb1 also controls cellular
differentiation by regulating expression of tissue-specific
transcription factors such as Myod1, via its association with
chromatin modifiers such as histone deacetylase, PCAF (P300/CBPassociated factor) and P300.
Pten
The Pten gene product is a lipid phosphatase that dephosphorylates
phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] at the 3
position. Pten is thus an antagonist of phosphatidylinositol-3-kinase
(PI-3 kinase), the activation of which regulates Akt/protein kinase B
(Akt/PKB). PI-3 kinase signaling also interacts with the p53 pathway,
activating the p53 inhibitor Mdm2. Pten therefore promotes p53
activity. PTEN deficiency, and the consequent overactivation of the
PI-3 kinase pathway, results in a hyperproliferative state and
increased cellular survival.
Hippo signaling
The Hippo pathway is a kinase cascade whose members link cellcell contact and cytoskeletal information to the downstream
activation of gene expression that regulates cell proliferation and
survival. The cytoskeletal proteins Merlin (Nf2) and Expanded
indirectly stimulate the phosphorylation of the protein Warts (also
known as Lats1) by Hippo (Mst1/2). Warts, in turn, interacts with,
phosphorylates and inactivates Yap1/Taz, a transcriptional coactivator that induces expression of genes related to proliferation
and survival. Yap1 interacts with the p53 family by potentiating
p73-mediated apoptosis.
induced tumors, whereas the non-regenerative ventral iris is
susceptible (Okamoto, 1997). These findings suggest that
regenerating tissues can resist or suppress carcinogen-induced
malignant transformation and/or propagation of tumor cells within
them. Although there is some evidence that urodeles may have a
lower incidence of cancer than adult anuran amphibians (Anver,
1992), the ability to enforce (albeit abnormal) tissue morphogenesis
over unrestrained proliferation and tumor formation seems to be a
specific property of the actively regenerating tissue and the
regeneration microenvironment, rather than a general property of
the organism as a whole, which is susceptible (Braun, 1965;
Zilakos et al., 1996; Oviedo and Beane, 2009). Urodeles, in
contrast to zebrafish, have not yet been used as a genetic
experimental organism for studying cancer. Now that genetic
experiments can be performed in axolotl, it will be interesting to
see the phenotypic outcome of genetic manipulation of oncogene
and tumor suppressor orthologs within and separate from the
regeneration environment.
In summary, examination of tumor susceptibility among
vertebrates does not clearly contradict or suggest that the continued
evolution of tumor suppressor genes and pathways has affected
regenerative capacity in a unified, predictable manner. What
available data in regenerative vertebrates strongly suggest is: (1)
that the extensive proliferative potency of cells within tissues
undergoing epimorphic regeneration can be controlled, suppressed
and switched off by powerful microenvironmental and or intrinsic
signals; and (2) that tumor formation is not a frequent outcome
during regeneration-associated proliferation, but is an event to
which regenerative vertebrates are susceptible, like other
vertebrates. A deeper understanding of the mechanisms controlling
cellular proliferation and differentiation (discussed below) at play
in both tumorigenesis and regeneration will be informative for
understanding both processes in different organisms.
Conservation of core tumor suppressor pathways
The four core tumor suppressor pathways we consider here (p53,
retinoblastoma, Pten and Hippo) are evolutionarily ancient and
exist in both highly and poorly regenerative organisms. By
definition, tumor suppressor pathways contain one or more tumor
suppressor genes, each defined by the characteristics of undergoing
biallelic inactivation in tumors, conferring tumor susceptibility in
individuals who inherit a single mutant allele, and being inactivated
in sporadic tumors (Sherr, 2004). Discussions of each of these
pathways are usually weighted towards a focus on tumor
suppressor aspects, largely because mutated alleles of these genes
were first identified in individuals with tumor syndromes, in tumors
themselves or in mutational loss-of-function screens that yield
tumor phenotypes. However, it is now understood that the core
tumor suppressor pathways are also key regulators of many
processes related to, but distinct from, cancer prevention. It is
generally accepted that it is these other functions that are most
conserved and are the targets of the selective pressures that drove
their evolution.
p53
The prototypical tumor suppressor gene and pathway is p53 (Box
1, Fig. 2), and virtually all cancers either harbor mutations in the
p53 gene or have functionally inactivated p53 via other key
pathway components (Junttila and Evan, 2009). Three p53 family
members exist in fish, amphibians and mammals: p53, p63 (Trp63)
and p73 (Trp73) (Lu and Abrams, 2006), although to date only one
p53 member has been identified in urodeles (Villiard et al., 2007).
Although all three can each induce growth arrest and apoptosis,
p53 is the primary tumor suppressor. Unlike p63 and p73, it is
frequently mutated in human and mouse cancers, and results in a
tumor prone phenotype when depleted from or disrupted in the
germline (Malkin et al., 1990; Donehower et al., 1992).
Surprisingly, p53 is the newest family member: the ancestral
member in single cell choanoflagellates and metazoan sea
anemones is more closely related to p63 and p73 (Belyi et al.,
2010). No p53 family members have been identified in prokaryotes
or yeast. It has been proposed that the evolutionary appearance of
p53 as distinct from the primitive p63/p73-like ancestor coincides
with the emergence of adult somatic tissue stem cells in which it
functions as a tumor suppressor by policing DNA damage (Belyi
et al., 2010), whereas the ancestral gene in hydra, nematodes and
DEVELOPMENT
Box 1. Cellular functions of key tumor suppressors
Development 140 (12)
HYPOTHESIS 2505
Development 140 (12)
p16ink4A
p130
P
p107
PIP2
pRb1
P
E2F Repression
Lats1/2
Mer
pRb1
Mst1/2
P
Arf
Exp
Lats1/2
P
Yap1
P
P
P
PIP3
E2F Activation
Activation
Yap1
PI-3K
Pten
CDK
Akt/PKB
P
p21Cip1
Mdm2
P
GSK
mTOR
Foxo
14-3-3
Bax
p53
p63
Puma
Bak1
p73
flies functions to maintain genomic fidelity in the germline (Belyi
et al., 2010; Dötsch et al., 2010). Notably, p53-null flies do not
develop tumors (Kondo, 1998; Sogame et al., 2003). In planaria, a
highly regenerative multicellular organism, one p53 ortholog has
been identified and shown to have important functions in stem cell
self-renewal and differentiation, as in vertebrates (Pearson and
Sánchez Alvarado, 2010). In vertebrates, p63 and p73 perform
distinct functions in embryogenesis of the skin and appendages,
and the immune and nervous systems, respectively. In addition,
they retain the ancestral role of ensuring germ cell fidelity (Suh et
al., 2006; Tomasini et al., 2008). Thus, the ancient germline
protective function of p53 orthologs is conserved, and the newer
tumor suppressor function that is the principal function of p53
exists in multicellular creatures that have adult tissue stem cells,
including planaria, and both highly and poorly regenerative
vertebrates (Villiard et al., 2007) (Lu and Abrams, 2006; Belyi et
al., 2010).
Retinoblastoma proteins
The retinoblastoma proteins pRb1 (p105), p107 and p130 are
central to regulation of the G1/S transition of the cell cycle (Box
1, Fig. 2), and are at the focal point of the central growth control
retinoblastoma pathway. Like p53, Rb1 (the gene encoding pRb1)
has two other family members in mammalian cells, p107 (Rbl1)
and p130 (Rbl2) (Ewen et al., 1991; Hannon et al., 1993; Li et al.,
1993). The retinoblastoma pathway is also largely conserved in
eukaryote evolution and homologs exist in plants and animals.
However, the ancestral member of this family appears to be Rb1,
because, at the amino acid level, nematode and Drosophila
homologs most closely resemble pRb1. No retinoblastoma
protein relatives have been identified in unicellular organisms.
Human RB1 was first identified as a tumor suppressor mutated in
children who develop retinal tumors (Knudson, 1971; Cavenee et
al., 1983; Friend et al., 1986). However, it was subsequently
found to have broad functions in promoting tissue differentiation
and maturation during embryogenesis. Deletion of Rb1 in the
mouse germline results in an embryonic lethal phenotype that is
characterized by defects in development of the CNS,
hematopoiesis and skeletal muscle (Clarke et al., 1992; Jacks et
al., 1992; Lee et al., 1992). Rb1 deficiency results in
inappropriate cycling of cells and failure of terminal
differentiation and expression of mature tissue specific gene
expression profiles. The ‘pocket domain’ (that mediates binding
to other proteins) of pRb1 is highly conserved in vertebrates (Lee
et al., 1998). Its biochemical functions are also conserved in
zebrafish and urodeles, although the role of pRb1 as a tumor
DEVELOPMENT
Fig. 2. Molecular functions and intracellular networks of tumor suppressor pathways. The principal components of the Hippo (green),
retinoblastoma (blue), p53 (red) and Pten (purple) tumor suppressor pathways. The four primary tumor suppressor genes [Mst1/2 (Hippo), Rb1, p53 and
Pten] are octagons with bold outlines. Other tumor suppressors within these pathways are octagons without bold outlines. Additional retinoblastoma
and p53 family members (p107 and p130; p63 and p73, respectively) are shown, although their functions should not be considered redundant, and p63
and p73 are not well documented tumor suppressors. Broken lines indicate regulation at the level of gene expression. Solid lines indicate regulation by
other mechanisms. Yellow ovals represent effector molecules in affected pathways. Zigzags indicate the lipid chains of PIP2 and PIP3. The main proteins
and activities that are discussed in this article are included in the figure. However, additional intracellular functions exist for some of the molecules
represented and additional components exist within the pathways. ‘P’ indicates phosphorylated protein. Activation and repression refer to
transcriptional regulation. Akt/PKB, protein kinase B; Arf, alternative reading frame product of Cdkn2a; Bak1, Bcl2-antagonist/killer 1; Bax, Bcl2-associated
X protein; CDK, cyclin-dependent kinase; Exp, Expanded; Foxo, O class of forkhead box proteins; GSK, glycogen synthase kinase; Lats1/2, large tumor
suppressor 1/2; Mdm2, transformed mouse 3T3 cell double minute 2; Mer, Merlin; mTOR, mammalian target of rapamycin; p16Ink4a, cyclin-dependent
kinase inhibitor 2 (Cdkn2a); p21Cip1, cyclin-dependent kinase inhibitor 1 (Cdkn1a); PI-3K, phosphatidylinositide-3-kinase; PIP2, phosphatidylinositol
bisphosphate; PIP3, phosphatidylinositol triphosphate; Puma, Bcl2 binding component 3 (Bbc3); Yap1, Yes-associated protein (Yorkie in Drosophila).
suppressor has not been studied in these species (Tanaka et al.,
1997; Thitoff et al., 2003; Ríos et al., 2011; Gyda et al., 2012).
Although inactivation of the retinoblastoma pathway is a
prerequisite for tumor formation, the crucial role of Rb1 in
forming normal tissues suggests that evolutionary selection has
probably operated primarily on its roles during embryogenesis,
rather than on its postnatal tumor suppressor functions.
Conversely, a key modulator of pRb1, the cyclin-dependent
kinase inhibitor Ink4a (Cdkn2a), is a mammalian tumor
suppressor that is frequently mutated in cancers, but has no
crucial role in development, as evidenced by the viability, fertility
and lack of structural defects in most tissues of Ink4a-null mice
(Serrano et al., 1996).
Pten
Pten is another tumor suppressor gene very commonly mutated in
human cancers (Li et al., 1997; Steck et al., 1997). A negative
regulator of PI-3 kinase signaling, Pten constrains cell size, number
and survival (Box 1, Fig. 2). Pten is essential during development
and Pten deficiency results in embryonic lethality at day 8.5 in
mice (Di Cristofano et al., 1998; Sun et al., 1999). Heterozygous
mice or mice conditionally mutant for Pten develop hyperplasia,
and exhibit reduced apoptosis and overgrowth phenotypes in
multiple tissues and organs. Similar phenotypes are also observed
upon mutation of the Drosophila, nematode, planaria, Xenopus and
zebrafish Pten homologs (reviewed by Cid et al., 2008). Pten has
not been identified and functions have not yet been studied in
urodele amphibians. The zebrafish and planaria genomes each
contain two Pten genes: in planaria, these appear to be fully
redundant, whereas the zebrafish paralogs have undergone some
functional divergence (Croushore et al., 2005; Faucherre et al.,
2008; Oviedo et al., 2008).
Hippo signaling
Among the newest major growth control pathways to be
identified is the Hippo signaling pathway (Box 1, Fig. 2), with
the major negative modulator of this pathway, the Hippo gene,
first discovered in 2003 in Drosophila (Wu et al., 2003) – the
species in which this pathway has been best characterized.
Although Hippo was discovered in ‘tumor suppressor’ screens in
Drosophila, the selected phenotypes are for overgrowths and
tumor-like formations, not for invasive cancer. However,
inactivating mutations in the upstream regulator Merlin
(neurofibromatosis 2; Nf2) lead to Schwann cell tumors and
meningiomas in humans. And the downstream effector of the
pathway, Yap1 (yes-associated protein 1) is an oncogene that is
frequently overexpressed in a wide spectrum of human cancers
and that induces hepatocellular carcinomas when overexpressed
in mice (reviewed by Pan, 2010). Available evidence indicates
that the tumor suppressor activity of the pathway is perhaps less
pervasive than that of p53, pRb1 and Pten, and that the function
of Hippo in organ size control and other aspects of development
predominate. The fundamental roles of Hippo signaling in
Drosophila development, first recognized in the restriction of
growth by Hippo of the imaginal discs (the epithelial primordia
of adult structures), are broad and affect many tissues and organ
systems, including the ovarian follicle and the CNS. The essential
cellular functions of Hippo signaling include coordination of the
transition from proliferative to postmitotic states and full
expression of the differentiated phenotype (reviewed by Pan,
2010). In zebrafish and Xenopus, Yap1 is expressed ubiquitously
during embryogenesis (Jiang et al., 2009; Nejigane et al., 2011),
Development 140 (12)
and in zebrafish it is required for development of the brain and
craniofacial structures (Jiang et al., 2009).
Recent innovations in tumor suppressor pathways might
alter regenerative capacity
As discussed above, the core tumor suppressor pathways are highly
conserved throughout evolution and are present in both highly and
poorly regenerative species. Although genomic information is
limited for urodeles, retinoblastoma proteins and p53 have been
identified, and their protein products have been confirmed to
function in at least some respects like their counterparts in other
species. Moreover, available information confirms similar, if not
completely conserved, core functions for the p53, retinoblastoma,
Pten and Hippo pathways in the other highly regenerative species,
including planaria and the vertebrate zebrafish. However, there are
potentially important differences in the pathways between highly
regenerative species and poorly regenerative higher vertebrates,
such as the diversification of the retinoblastoma and p53 pathways.
Moreover, when more sophisticated modulators of the pathways
are considered, key differences could underlie important
divergences among highly and poorly regenerative species. One
intriguing example is the evolutionary appearance of the Arf
(Cdkn2a) tumor suppressor (see Box 1 and Fig. 3) in nonregenerative vertebrates. As discussed above, the mammalian Ink4a
locus is a major tumor suppressor. In mammals, the Ink4a locus has
a highly unusual if not unique organization that results in the
production of two proteins, Ink4a and Arf, via separate first exons
and common second and third exons (Quelle et al., 1995). The two
proteins are encoded by alternative reading frames and have no
amino acid homology and have completely distinct biochemical
functions, but both are potent tumor suppressors in mice and
humans. Whereas homologs of Ink4a (a component of the
retinoblastoma pathway) exist in fish, amphibians and lower
metazoans, the earliest Arf (a component of the p53 pathway)
ancestor arose in birds (Gilley and Fried, 2001; Kim et al., 2003;
del Arroyo and Peters, 2005). Potential implications relevant to
regeneration have been proposed and are discussed below.
As another example, the p53 target p21 (Cdkn1a) acts as a
negative regulator of cyclin-dependent kinase activity. The p21
gene does not appear to exist in planaria (Pearson and Sánchez
Alvarado, 2010) but is present in zebrafish (Langheinrich et al.,
2002). p21 has been implicated in suppression of epimorphic
regenerative capacity in a mouse ear hole model (Bedelbaeva et al.,
2010), whereas deficiency of p53 had no apparent effect (Arthur et
al., 2010). Although the existence of p21 in zebrafish demonstrates
that potent regeneration can occur in organsisms that contain this
gene, it is possible that differences in regulation of p21 expression,
e.g. the presence or absence of Arf (see Box 1), or in Tgfβ/Smad
activity (Pardali et al., 2000) could limit expression of p21 during
regeneration in zebrafish. This possibility has yet to be tested
experimentally. Undoubtedly, other evolutionarily divergent genes
will be identified that modulate the core pathways in important
ways, and presumably have evolved to perform functions uniquely
important in the species in which they exist. Whether they also
result in physiological side effects that alter other functions such as
regeneration remains to be tested.
Core tumor suppressor pathways are required for
tissue regeneration
Unlike cellular proliferation that replenishes the hematopoietic
system or re-epithelializes skin wounds, regeneration of other
complex tissues and organs after injury involves coordinated
DEVELOPMENT
2506 HYPOTHESIS
A Lower vertebrates
Ink4a
pRb1
Mdm2
Differentiation
E2F Repression
CDK
p53
p53
pRb1
Growth arrest
Apoptosis
Proliferation
P
E2F Activation
B Mammals
Ink4a
locus
Arf
p16Ink4A
pRb1
Mdm2
CDK
p53
Differentiation
E2F Repression
pRb1
Proliferation
P
Growth arrest
Apoptosis
E2F Activation
Fig. 3. Evolution of the Ink4a tumor suppressor locus in vertebrates.
(A,B) The Ink4a locus is a good example of a tumor suppressor locus that
has evolved to differ significantly between lower vertebrates (A) and
mammals (B). Homologs of the cyclin-dependent kinase inhibitor
p16Ink4a (Cdkn2a) exist throughout vertebrates, while the earliest known
ancestor of Arf (alternative reading frame) arose in chickens. In mammals,
the Ink4a locus uses distinct first exons and common second and third
exons to generate the two proteins, Arf and p16Ink4a, transcribed in
alternative reading frames, resulting in no amino acid homology.
P16Ink4a functions in the retinoblastoma pathway to inhibit the
phosphorylation of pRb1, thereby promoting sequestration of E2F
(shown in A). Arf functions primarily in the p53 pathway and inhibits
Mdm2, resulting in stabilization of p53 and enhancement of p53
functions. Arf can be induced directly by E2F, resulting in the arrest of
cells that have reached a threshold of proliferation signaling through the
pRb1/E2F pathway. Broken lines indicate regulation at the level of gene
expression. Solid lines indicate regulation by other mechanisms. Primary
tumor suppressor genes are in bold octagons. Yellow ovals represent
effector molecules in affected pathways. CDK, cyclin-dependent kinase;
Mdm2, transformed mouse 3T3 cell double minute 2; P, phosphorylation.
generation and patterning of multiple different cell types and
structures simultaneously. Because of this complexity, it is
challenging to envision a scenario whereby this ability evolved
independently to accomplish normal development and
regeneration. A more parsimonious possibility that is becoming
increasingly well supported is that regeneration of complex tissues
and organs involves many of the same programs and machinery
that are used during development (reviewed by Mariani, 2010;
Nacu and Tanaka, 2011). Therefore, it would follow that the core
tumor suppressor pathways should recapitulate their important
developmental functions during regeneration. However, it is also
clear that regeneration involves certain processes that are distinct
from embryonic development, such as the initial wound healing
step, and activation of quiescent progenitors or reassignment of
terminally differentiated cells to execute the regenerative process.
Tumor suppressors could have regeneration-specific functions in
these processes.
General physiological processes are frequently recapitulated in
regeneration, and the specific activities of tumor suppressors may
be similarly conserved. One example of this phenomenon is
provided by the role of p53 in the injury response in Drosophila
imaginal discs. Imaginal discs undergo regeneration after injury, in
a process involving compensatory proliferation that is stimulated
by injured, dying or dead cells (reviewed by Worley et al., 2012).
The proliferative response is the end result of a multistep process
that first requires cell cycle arrest, autocrine and paracrine signaling
[including induction of the Wingless (Wg) pathway]. Interference
with any of these steps has been shown to block compensatory
proliferation, and dp53 function is crucial for regeneration, as dp53
mutants fail to induce G2 arrest, wg expression or compensatory
proliferation (Wells et al., 2006). Thus, in this context, p53 appears
to promote rather than restrain proliferation. The authors of that
study suggest that these dp53 functions, which appear to be at odds
with tumor suppressor functions of mammalian p53, may relate
more closely to functions that have been adopted by p63 during
vertebrate epidermal development (Wells et al., 2006). The
important role of p63 in promoting epidermal progenitor cell
renewal (Koster et al., 2004) suggests that parallels exist between
mechanisms of p53 family functions in regeneration and uninjured
tissue formation and renewal.
That tumor suppressors would be involved in regulating the
proliferation-dependent aspects of regeneration is to be expected
given their known molecular functions, although the example above
suggests that even in this context, their roles may be more complex
than might be anticipated. However, there is also evidence for roles
of tumor suppressors in regulating regeneration independently of
proliferation, notably in the case of peripheral nerve axon
regeneration. Given the nerve dependence of regeneration of limbs
and other tissues (Singer, 1974), this is particularly important to the
biology of regeneration of complex tissues. p53 was found to be
necessary for central and peripheral nerve regeneration, a function
not necessarily predicted from developmental studies of p53 (Di
Giovanni et al., 2006). As axon and dendrite regeneration occur in
the absence of neuronal cell division, the dependence on p53 for
neurite outgrowth in culture and peripheral nerve regeneration in vivo
in mice represents an unexpected activity of this tumor suppressor.
p53 targets activated in this context, coronin 1b (Coro1b) and Rab13,
are distinct from those involved in growth arrest or apoptosis.
Although CNS defects frequently occur in p53 knockout mice
(Armstrong et al., 1995), it is not yet clear whether these
developmental phenotypes are a result of the pro-apoptotic function
of p53, or whether they reflect an activity conserved between the
developmental and regeneration contexts.
The only highly regenerative multicellular organism in which
the role of Pten has been examined in the context of tissue
regeneration is planaria. The mechanisms of regeneration in
planaria differ significantly from those in vertebrates, primarily
owing to the existence of a pluripotent cell population, the
neoblasts, dispersed throughout the body of the adult flatworm
(Newmark and Sánchez Alvarado, 2000; Newmark and Sánchez
Alvarado, 2002; Reddien and Sánchez Alvarado, 2004) – no
equivalent of which exists in vertebrates. However, as in the
DEVELOPMENT
HYPOTHESIS 2507
Development 140 (12)
2508 HYPOTHESIS
Putative regeneration-promoting functions of
tumor suppressors
Considering the known tumor suppressor activities of p53, pRb1,
Pten and Hippo generates some hypotheses for how these genes
and pathways might be predicted to function to support
regeneration (Fig. 1).
Tumor suppression
First, it is easy to speculate that tumor suppressors would suppress
tumorigenesis during the periods of extensive and rapid cellular
proliferation that occur during regeneration. For pathways such as
Hippo and Pten, one could predict that the known function of organ
size regulation would be harnessed to restrain and organize tissue
growth during regenerative re-growth – preventing tumor
formation. For p53 and pRb1, the classical tumor suppressor
functions of preventing inappropriate proliferation and eliminating
or enabling repair of stressed or genome-damaged cells would be
expected to be important for ensuring a healthy and tumor-free
regenerate. In mammals, the occurrence of carcinomas in
chronically regenerating liver and skin presumably represent rare
failures of these functions in the setting of the long-term presence
of proliferative signals. Although the absolute numbers of cell
divisions involved in newt limb regeneration do not approach those
seen during normal turnover of mammalian skin occurring over a
lifetime, it is still remarkable that a newt can successfully
regenerate amputated limbs many times, each involving massive
proliferation over a relatively short period. That newt limb
regeneration proceeds without tumor formation, implies that potent
tumor suppressive functions must be involved during the
regenerative process.
Promotion of differentiation
The tumor suppressor genes discussed in this Hypothesis have all
been shown to promote differentiation – a potent anti-cancer
activity – in various contexts, and this would have an obvious role
during regeneration. Just as crucial for regeneration as the
formation of a highly proliferative blastema is the process of
inducing cell cycle exit and the expression of genes that
characterize the mature differentiated phenotype. As mentioned
above, Rb1 is the classic example of a gene that performs this
function (Pajcini et al., 2010). Experiments that disrupt
retinoblastoma protein function in a regenerative setting during or
after the blastema stage have not yet been performed but would be
important to demonstrate the importance of this function and its
conservation from embryogenesis to regeneration.
Induction of apoptosis
Induction of apoptosis is a central function of p53 in tumor
suppression (Vousden and Prives, 2009) and can also be attributed
to other tumor suppressors in certain contexts (Basu et al., 2003;
Lehman et al., 2011). Apoptotic activity is particularly prominent
during limb formation, to separate the digits by eliminating the
interposed epidermal and mesenchymal tissue (Zuzarte-Luis and
Hurle, 2005). This raises the issue of whether the classical tumor
suppressor pathways are involved in this process. There is no
evidence to date of a role for tumor suppressor pathways in
regulating interdigit programmed cell death, either in
development or regeneration, but the apoptotic machinery
downstream of p53 family members [Bax, Bak1, Bid, Bim
(Bcl2l11) and Puma (Bbc3)] is known to control this process
(Lindsten et al., 2000; Ren et al., 2010). Although it has long
been known that interdigit programmed cell death is mediated by
BMP signaling (Hernández-Martínez and Covarrubias, 2011), the
immediate upstream communication to the Bcl2 family in such
cases is unclear. Normal limbs and digits form in p53-deficient
mice, but p63 may be a better candidate for regulating apoptosis
in this context, as it has important epithelial and appendage
developmental roles and can activate Bax-dependent apoptosis in
other contexts (Gressner et al., 2005). Moreover, Pten and Hippo
can cooperate with p53 family members (p73 in this case) to
regulate apoptosis (Basu et al., 2003; Lehman et al., 2011). Limb
development and regeneration therefore provides an ideal setting
DEVELOPMENT
highly regenerative teleosts and urodeles, planaria regeneration
involves the formation of a blastema (an epithelium-covered mass
of proliferating mesenchymal cells that will undergo
morphogenesis and patterning during regeneration) and although
these are not fully equivalent structures, the molecular and
cellular processes controlling proliferation and differentiation
may share significant similarities with those occurring during
vertebrate regeneration. Amputation experiments in planaria in
which the two Smed-Pten genes were suppressed using RNAi
resulted in inappropriate proliferation of neoblasts, failure of both
differentiation and tissue regeneration, and the production of
abnormal tissue outgrowths (Oviedo et al., 2008). This striking
example underscores the importance of limiting proliferative
responses during regeneration and demonstrates a necessary
function of Smed-Pten in forming normal tissues during
regeneration. Although the role of Pten has not yet been
examined in vertebrate epimorphic regeneration, a very different
role for this tumor suppressor has been identified in mammalian
peripheral and central nerve regeneration (Park et al., 2008; Liu
et al., 2010; Sun et al., 2011) (discussed below).
As in the case of Pten, one would predict that loss-of-function
experiments targeting Rb1 or Hippo would also impair regeneration
of complex tissues by disrupting patterning or differentiation – as
both factors regulate these processes during development. There is
evidence from Drosophila and mouse studies that the Hippo
pathway is a regulator of intestinal, epidermal and neural
progenitor function. Moreover, after intestinal or liver injury,
components of the Hippo pathway are important regulators of
repair (reviewed by Zhao et al., 2011). The crucial role of Rb1 in
the transition from a proliferative state to a postmitiotic
differentiated one has been amply demonstrated in developmental
studies in mice (Burkhart and Sage, 2008) and in studies of muscle
cells in culture (Gu et al., 1993; Lassar and Münsterberg, 1994;
Novitch et al., 1996). The failure of organized hematopoiesis,
neurogenesis and myogenesis in the absence of Rb1 (Clarke et al.,
1992; Jacks et al., 1992; Lee et al., 1992) strongly suggests that
similar outcomes would be observed if Rb1 were rendered deficient
during tissue regeneration. Moreover, cell culture experiments in
which suppression of Rb1 was used to induce muscle cell
proliferation required re-establishment of Rb1 function to induce
differentiation of myoblasts and incorporation into muscle (Pajcini
et al., 2010).
Considering the significant use of developmental programs
during tissue regeneration, together with the experimental
regeneration data available to date, leads us to conclude that tumor
suppressor function is a likely requirement for regeneration of
complex tissues. Some of these requirements relate to the known
functions of tumor suppressors in orchestrating proliferation,
differentiation and cell death, whereas newly recognized functions,
such as the promotion of axon regeneration by p53, are also being
revealed.
Development 140 (12)
in which to analyze the potential pro-apoptotic functions of tumor
suppressors during regeneration.
Metabolic regulation
Finally, another potentially important possible function of p53
during regeneration relates to its more recently recognized roles in
metabolism – primarily discussed in the context of tumorigenesis
(reviewed by Gottlieb and Vousden, 2010). Under conditions of
daily life and during stress, rather than eliminate cells, p53
functions to lower reactive oxygen species (ROS) levels and to
promote DNA repair and cellular survival. After injury, oxygen,
lactate and ROS levels are drivers of healing and regeneration
(Trabold et al., 2003; Thom, 2009), and it has been shown that tight
regulation of elevated ROS is crucial for tail regeneration in
Xenopus (Love et al., 2013). As such, the regeneration milieu may
be considered a ‘low stress’ environment in which p53 could
function to promote survival (rather than trigger apoptosis) (Fig. 1).
Whether cells in the regenerate undergo a type of metabolic
transformation similar to that seen in cancer (Gottlieb and Vousden,
2010) remains to be seen. Although experimental evidence is
currently lacking, it is tempting to speculate that p53 could promote
cellular survival and maintenance during regeneration as it can in
cancer (Kim et al., 2009).
Putative ‘regeneration suppressor’ roles for tumor
suppressors and potential exploitation in
regenerative medicine
Although solid tissue regeneration requires the restoration of
architecture and the interaction between different cell types, in vitro
studies have primarily focused on cellular regeneration or
reprogramming coupled to proliferation to produce copies of a
single cell type. Conceptually translating in vitro properties to
reflect in vivo functions carries significant caveats, but in vitro data
have formed the basis of many tumor suppressor studies,
corroborating and, in some cases, presaging in vivo findings.
Distinct growth properties in vitro have also been identified in cells
from highly regenerative vertebrates that could signify important
differences from mammalian cells that may impact regeneration in
vivo. For example, urodele blastema cells can replicate indefinitely
without senescence in culture (Ferretti and Brockes, 1988), and
cellular senescence in the regenerative zebrafish is much slower
than in mammals (Kishi et al., 2003). As senescence signals in
mammalian cells are transmitted and executed through the
retinoblastoma and p53 pathways (Ben-Porath and Weinberg,
2005), differences in retinoblastoma protein and p53 regulation
could be expected to influence distinct senescence phenotypes of
highly versus poorly regenerative species, an issue that could easily
be addressed in cell culture experiments.
Understanding how cell growth and division are controlled in
cultured cells may inform regeneration studies in two broad ways.
First and most direct is the potential to use this knowledge to
improve cell-based approaches in regenerative medicine, where the
emphasis lies in the ability to produce desired cell types in
abundance. Although the importance of tumor suppressor genes in
stem cells, especially in relation to aging, has been well studied and
reviewed elsewhere (Sharpless and DePinho, 2007), other
examples are illustrative here. For example, the derivation of
induced pluripotent stem cells (iPSCs) from differentiated somatic
cells, a process that requires extensive proliferation and extensive
reprogramming of gene expression, must overcome the significant
roadblock of in vitro senescence. Senescence in this context, as in
others, is induced by the core tumor suppressor pathways, and,
HYPOTHESIS 2509
conversely, suppression of tumor suppressor function can
significantly increase the yield of iPSC colonies – as has been
shown for Ink4a/Arf and p53. Although iPSC colonies proliferate
indefinitely once they are formed, bypassing or overcoming
Ink4a/Arf and p53 function appears to be a crucial step to gain
proliferative capacity (Hong et al., 2009; Kawamura et al., 2009;
Li et al., 2009; Marión et al., 2009; Utikal et al., 2009). Another
approach under investigation is the expansion of committed cell
populations by transient interference with tumor suppressor
function, thereby conferring growth capacity while retaining the
original identity (Blau and Pomerantz, 2011). By contrast, a
potential hazard of in vitro reprogramming and culture expansion
approaches is genomic instability and potential induction of
tumorigenicity, and increased dosage of tumor suppressor genes
may reduce this danger (Menendez et al., 2012).
Second, comparing differences in evolved tumor suppressor
genes and pathways among species may shed light on differences
in intrinsic in vivo regenerative capacities. The study of the process
of cellular dedifferentiation in newts identified the retinoblastoma
pathway as a key regulator (Tanaka et al., 1997), and this
mechanistic understanding, coupled with the absence of the Arf
tumor suppressor in highly regenerative species, led to experiments
resulting in the ability to mimic newt cellular behaviors in
mammalian muscle cells (Pajcini et al., 2010). Whether the absence
of ARF in highly regenerative vertebrates contributes to their
increased potential in vivo is currently being examined.
Finally, breakthroughs with direct clinical implications have
been made in targeting tumor suppressors in vivo to enhance axonal
regeneration by mechanisms distinct from effects on proliferation.
A number of studies have demonstrated that axon regeneration of
central and peripheral neurons can be significantly enhanced by
interfering with the expression of Pten after crush injuries (Park et
al., 2008; Liu et al., 2010; Sun et al., 2011). Interestingly, targeting
of p53 in similar experiments promoted neuronal survival but did
not enhance regeneration (Park et al., 2008). These remarkable
findings in mice established an important link between control of
protein synthesis by the Pten pathway and regeneration, a link not
easily predictable in the context of canonical PTEN tumor
suppressor functions.
Conclusion
The diverse functions of the tumor suppressors discussed in this
Hypothesis article defy the simple model that tumor suppressors
restrict proliferation, are therefore anti-regeneration, and can be
bluntly targeted to enhance regeneration. Instead, from the
available regeneration data reviewed here along with known
functions in relevant contexts such as development and postnatal
metabolic regulation, we conclude that tumor suppressors in
general support regenerative processes. This view holds both in
terms of tumor suppressor roles in modulating proliferation and
differentiation, as well as in protecting cellular genomic integrity
that is likely at risk during periods of metabolic and proliferative
stress. In fact, enhancing tumor suppressor function in the right
temporal context may be important in future efforts to induce
regenerative processes. However, it is also clear in the case of Arf
(which does not exist in highly regenerative vertebrates such as
fish and amphibian) that some key tumor suppressor genes are
not required for efficient regeneration without malignant
transformation. Moreover, the existence of multiple family
members, as with p53 and retinoblastoma genes, may allow
certain family members to have taken on specific roles during
regeneration. These observations could indicate opportunities to
DEVELOPMENT
Development 140 (12)
selectively modify tumor suppressor activities to improve desired
regenerative outcomes. Indeed, the experiments described in this
article support this view and have already shown that inhibition
of tumor suppressor function can productively permit
dedifferentiation, reprogramming and expansion of desired cell
types.
It remains to be seen whether perturbation of tumor suppressor
function can enhance regeneration of complex tissues in vivo, as
has been shown for regeneration of somatic stem cells in the
hematopoietic system, endocrine cells of aged animals (Janzen et
al., 2006; Krishnamurthy et al., 2006) and axons (Park et al., 2008;
Liu et al., 2010; Sun et al., 2011). Additional loss-of-function
experiments in mammals may resolve this. In zebrafish and
salamanders, the introduction of mammalian tumor suppressors
such as Arf during regeneration will determine whether they harbor
true regeneration suppressor activity. In the context of in vitro
cellular regeneration protocols, the transient targeting of select
tumor suppressors during windows of opportunity may eventually
elicit expansion without negative consequences. A sophisticated
understanding of the complex functions of tumor suppressors in
distinct contexts is merited and will most certainly continue to be
informative for regenerative medicine applications and basic
questions alike.
Acknowledgements
The authors apologize to colleagues whose important work was not cited
owing to space limitations. We would like to thank Robert Hesse and other
members of our laboratories for insightful comments on the manuscript.
Funding
J.H.P. is supported in part by funds from the University of California San
Francisco Departments of Surgery and Orofacial Sciences; the University of
California San Francisco Program for Breakthrough Biomedical Research
Opportunity Award; and a California Institute of Regenerative Medicine (CIRM)
New Faculty Physician Scientist Award. H.M.B. is supported by National
Institutes of Health (NIH) grants; a subcontract from the University of
Maryland, Baltimore on an NIH grant; an NIH Director’s Transformative
Research Award; a CIRM grant; and the Baxter Foundation. Deposited in PMC
for release after 12 months.
Competing interests statement
J.H.P. and H.M.B are scientific founders of Didimi, Inc.
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