In vivo modeling of tumor suppressor p53 functions and regulation has a history of
unexpected and even enigmatic outcomes [1], despite the status of p53 as the most
frequently mutated gene or dysfunctional pathway in human cancers [2], [3]. Beginning
with the surprising viability of the first mice deleted for Trp53
[4], [5], various hypotheses of compensation, cell type–specificity, stimulus-dependent
response, or modifier influences were posed to explain how an exquisitely regulated
transcription factor, implicated in a vast array of pathways [6], appeared to have
no impact on development. Limited background-specific developmental and fertility
problems do occur, especially in female p53-null mice [7], [8], and deletion of potentially
compensatory p53 family members, p63 and p73 isoforms, leads to profound developmental
and tissue-specific phenotypes [9], [10]. But overall, the most striking result of
p53 loss in vivo is early tumor predisposition in p53−/− mice, which lack genomic
surveillance provided by p53-mediated regulation of cell cycle arrest, apoptosis,
and senescence.
As reported by Concepcion et al. in this issue of PLoS Genetics
[11], expectations built on cell-based studies of p53 response are again unrealized
in mouse models. Previously, multiple in vitro analyses suggested that microRNA (miR)-34
family members are important players in a p53-regulated network of genomic surveillance
[12]–[17] (Table 1). Together, these studies strongly supported the view that p53
response to multiple stimuli depended on miR-34, and that ectopic expression of miR-34
was sufficient to elicit p53 response, consistent with miR-34 functioning as a bonafide
tumor suppressor. However, Concepcion et al. report that complete inactivation of
the entire family of miR-34 genes (miR-34a/b/c) or knockout of each individual miR-34
gene in mice leads to little or no change in p53-mediated functions in tumor suppression
[11].
10.1371/journal.pgen.1002859.t001
Table 1
A list of different in vitro and in vivo model systems used to study miR-34 functions.
Model System
Description
miR-34
Functional Outcome
Ref.
mESCs
Mouse embryonic stem cells
Genetrap-mediated deletion
Decreased spontaneous apoptosis during differentiation
[12]
NSCLCs
Non-small cell lung cancer cells
Overexpression
Inhibits growth
[12]
SW480
p53 mutant colon cancer cells
Overexpression
G1-arrest
[12]
Wi38
Human diploid fibroblasts
Depletion
Protection from Staurosporine-induced apoptosis
[12]
IMR90
Primary lung fibroblasts
Overexpression
Growth inhibition (G1 and G2 arrest), and senescence
[13]
A549
Human alveolar adenocarcinoma cells
Overexpression
G1-arrest
[13]
HCT116
Human colon cancer cells
Overexpression
G1-arrest
[13]
TOV21G
Human ovarian cancer cells
Overexpression
G1-arrest
[13]
MEFs
Mouse embryonic fibroblasts
Overexpression
Apoptosis
[13]
H1299
Human lung cancer cells
Overexpression
Reduced colony formation
[14]
U2OS
Human osteosarcoma cells
Depletion
Reduction in Etoposide-induced apoptosis
[14]
HCT116 (p53+/+ or p53−/−)
Human colon cancer cells
Long-term overexpression
Apoptosis
[15]
HCT116, RKO
Human colon cancer cells
Overexpression
Suppression of proliferation and induction of senescence
[16]
Mouse xenograft model
HCT116 or RKO cells were inoculated into nude mice
Subcutaneous administration of miR-34a/atelocollagen complexes
Suppression of cell proliferation and reduction in tumor volume
[16]
H1299
Human lung cancer cells
Overexpression
Apoptosis
[17]
U2OS
Human osteosarcoma cells
Overexpression
G1-arrest and reduction in colony formation
[17]
MiaPaCa2, BxPC3
p53 mutant human pancreatic cancer cell lines
Overexpression
Inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2 arrest;
sensitized the cells to chemotherapy and radiation
[34]
OSN1, OSN2
Neoplastic epithelial ovarian cells
Overexpression
Suppression of proliferation and reduced colony formation
[35]
Kelly, NGP
Neuroblastoma cells with MYCN amplification (+MNA)
Overexpression
Reduction in proliferation and increased apoptosis
[36]
SK-N-AS
Neuroblastoma cells without MYCN amplification (-MNA)
Overexpression
Reduction in proliferation and increased apoptosis
[36]
Mouse mir34a−/−; mir34b/c−/−
miR-34 knockout mouse in C57BL/6 background
Germline deletion of miR-34
Efficient reprogramming, no effect on proliferation
[30]
Mouse mir34TKO
miR-34 knockout mouse in129SvJae and C57BL/6 mixed background
Germline deletion of miR-34
Normal p53 activity
[11]
Interest in a miR-34 axis as mediator of p53-response begins with the niche that miRNAs
fill in regulation of RNA expression. miRNAs are small, regulatory non-coding RNAs
that generally mediate post-transcriptional silencing of a number of specific target
mRNAs [18]. More than 50% of human miRNA genes are found within cancer-associated
or fragile sites of the genome, which suggests that miRNAs play essential roles in
tumorigenesis [19]. The identification of miRNAs as regulatory targets of p53 [20]
suggested their potential involvement in tumor suppression, and expanded the repertoire
of p53 downstream targets to both coding and non-coding genes. Further, the view that
p53 both positively and negatively regulates gene expression could now rely on increased
expression of miRNAs as a mechanism for p53-mediated, indirect repression of gene
expression [13], [20], in addition to the few documented cases of direct repression
by p53 binding to chromatin [21]–[25].
The members of the evolutionarily conserved miR-34 family, which arise from three
different transcripts at two different gene loci in vertebrates, were the first of
several non-coding RNAs identified as directly activated by p53 in response to genotoxic
stress [13], [26]. miR-34a is at 1p36, a region commonly deleted in tumors, and miR-34b
and miR-34c share a common primary transcript arising from 11q23 [27], [28]. miR-34a,
b, and c are expressed at very low levels in several types of cancers [28]. Previous
reports show that p53 directly activates miR-34a/b/c expression and, dependent on
cellular context, they act downstream of p53 in mediating cell cycle arrest or apoptosis
[29]. The current list of validated miR-34 downstream targets includes several genes
that are repressed during cell cycle arrest or apoptosis when p53 is activated [28].
Given the rationale provided by these studies in cultured cells (Table 1), multiple
laboratories created genetic knockout models of either miR-34a or miR-34b/c, or a
compound mutant animal harboring homozygous deletion of all three miR-34 family members
(miR-34TKO) [11], [30]. Surprisingly, mice bearing the miR-34 deletion(s) developed
normally, are born at the expected Mendelian ratio, and are fertile [11]. The authors
subjected the mice and derived mouse embryonic fibroblasts (MEFs) to a battery of
tests to assess any impact on p53-dependent tumor suppression. MEFs obtained from
mir-34TKO mice have a slightly higher proliferation rate, but reach senescence with
kinetics similar to wild-type MEFs. In response to genotoxic threats, miR-34–deficient
MEFs are indistinguishable from wild type: they undergo p53-dependent cell cycle arrest
and apoptosis. With ectopic expression of oncogenic K-Ras, p53-deficient MEFs are
readily transformed, which is not true of K-Ras–expressing miR-34−/− MEFs.
In the intact mouse, the story is similar: aging cohorts of mir-34TKO mice remain
healthy with no spontaneous tumors, in contrast to p53-null mice [4]. In fact, miR-34–deficient
mice remain remarkably healthy and tumor-free for at least 60 weeks after irradiation.
Assays of apoptosis in response to irradiation proved positive in tissues of these
mice, which additionally exhibited no acceleration of tumor progression in Eμ-models
of B-cell lymphomagenesis. All of these assessments of p53 functions in vivo undermine
the view that miR-34 functions as a tumor suppressor or is an essential component
of the p53-tumor suppression network.
Although miR-34 proved nonessential in the most highly studied examples of p53 function
(senescence, cell cycle arrest, apoptosis, and tumor suppression), it remains possible
that miR-34 is involved in other p53-influenced processes, such as metabolism, autophagy,
stem cell quiescence, differentiation, and embryogenesis [6]. For example, specific
links between miR-34– and p53-regulated functions have been forged in stem cells [26].
miR-34–deficient MEFs are more efficiently reprogrammed to induced pluripotent stem
cells (iPSCs), by expression of pluripotency factors and c-myc [30], compared to wild-type
counterparts. While this study of miR-34 as a barrier to reprogramming does not establish
a direct tie to p53, it complements multiple reports that depletion of p53 or dysfunctional
p53 pathways enhance the efficiency of reprogramming differentiated, somatic cells
to iPSCs [31]. Recently, we showed that p53 promotes human embryonic stem cell differentiation
by direct activation of p21 and miRNAs, including miR-34a, which repress pluripotency
factors and SIRT1 [32]. Taken together, these results indicate that miR-34 has pro-differentiation
effects in maintenance of nontransformed, somatic cells, some of which are p53-dependent.
In the future, miR-34–deficient mouse models will be valuable in addressing whether
miR-34 functions downstream of p53 in a tissue- and/or context-specific manner. miR-34a,
miR-34b, and miR-34c share the same seed sequence and target the same RNAs, although
differences in target accessibility or binding affinities may dictate their effectiveness.
Genome-wide expression analysis may be needed to determine family member–specific
effects, such as the reported regulation of c-MYC by miR-34b/c and not miR-34a [33].
Questions of specificity in gene targets for each member of a miRNA family and potential
compensation by other miRNAs may be addressed by studies in these and other miRNA
mouse models, perhaps still under development. Non-coding RNAs are thought to act
in networks that impact diverse cellular pathways, suggesting considerable challenges
ahead in asking the right questions and understanding the functional significance
of these RNAs.