The emerging functions of the p53-miRNA network in stem cell biology

Abstract

The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.

p53 as an Important Tumor Suppressor in Somatic Cells

p53, one of the most important tumor suppressors, is mutated in 50% of human cancers.¹ Inactivation of the p53 pathway is even more prevalent than p53 mutation alone, since components of this pathway besides p53 are also frequently mutated in human cancer.² Consonantly, p53 acts as a tumor suppressor in mouse genetic models. p53-knockout mice develop spontaneous tumors,^3,4 and p53 deficiency strongly cooperates with other genetic lesions to accelerate tumorigenesis in various cancer models.⁵ Besides its well-characterized tumor suppressive function in regulating cell proliferation and apoptosis, p53 also impacts profoundly on diverse biological processes, such as autophagy, metabolism, ROS regulation and aging.^6-10

p53 functions mainly as a transcriptional factor activated in response to a variety of stimuli, such as DNA damage, hypoxia and oncogene expression.¹¹ Through different molecular mechanisms, these stimuli stabilize p53, leading to its accumulation and nuclear translocation. Upon translocation, p53 triggers expression of a network of downstream targets to elicit cell type- and context-dependent cellular responses, including cell cycle arrest, senescence, DNA repair and apoptosis.^2,9,10,12 These downstream effects collectively suppress tumor formation and maintain genomic stability by eliminating or repairing the damaged cells. In recent years, it has become increasingly clear that p53 acts as a global gene regulator to achieve these effects, both trans-activating target genes and downregulating a large number of genes, either directly or indirectly. In addition, p53 can function through transcription-independent mechanisms to regulate gene expression (see below).¹³

The p53 Network in Pluripotent Stem Cells

As the guardian of the genome, p53 regulates cell proliferation, survival and genomic stability not only in somatic cells, but also in pluripotent stem cells.^14,15 In somatic cells, p53 is destabilized but can be rapidly stabilized and activated in response to genotoxic stress and aberrant oncogene activation. Once acutely activated in somatic cells, the p53 pathway initiates cell cycle arrest, apoptosis, cellular senescence and DNA repair to maintain genomic integrity.^2,9,10,12 Interestingly, ES cells regulate and respond to p53 differently than these somatic cells. ES cells express higher levels of p53, but it is predominantly cytoplasmic.^16,17 Upon genotoxic stress, activated p53 translocates to the nucleus of ES cells through an unknown mechanism.¹⁷ Although debates still exist as to whether such translocated p53 can activate its canonical targets in ES cells,^14,16,17 the functional importance of basal p53 expression in ES cells is clearly demonstrated by the hyperproliferation, resistance to apoptosis and compromised genomic stability observed in p53-deficient human ES cells.^14,15

The unlimited self-renewal capacity and pluripotent differentiation potentials of ES cells pose unique circumstances for the p53 pathway to maintain their genomic stability. Interestingly, murine ES cells fail to undergo p53-dependent G₁/S cell cycle arrest or apoptosis in response to genotoxic stress.^18,19 p53, instead, triggers rapid ES differentiation, at least in part due to its repression of Nanog by recruitment of the corepressor mSin3A to the Nanog promoter.²⁰ p53 is activated and required for retinoic acid-induced differentiation in murine ES cells by the same mechanism,²⁰ since retinoic acid-treated ES cells exhibit increased transcription activity of p53, as demonstrated by the upregulation of mdm2 and the downregulation of Nanog.^20,21 By comparison, human ES cells undergo both p53-dependent and p53-independent apoptosis in response to genotoxic stress, and p53 activation also triggers rapid differentiation with concomitant decrease of Nanog and Oct4.²² Suppression of Nanog and Oct4, and possibly other pluripotency genes, by p53 is likely achieved through a combination of direct transcriptional repression by p53 and indirect post-transcriptional repression by p53-regulated miRNAs (see below). Furthermore, ES cells subjected to genotoxic stress exhibit rapid differentiation, which precedes the apoptotic response.²⁰ Such differentiation mechanisms effectively maintain genomic stability of the self-renewing ES cells. Like normal somatic cells, the differentiated lineage of ES cells undergoes p53-dependent cell cycle arrest or apoptosis in response to DNA damage, leaving the genetically intact ES cells alone to continue self-renewal within the population.

Paradoxically, despite its pro-differentiation function in ES cells, activated p53 also antagonizes differentiation by inducing multiple Wnt ligands.²³ Such Wnt induction by p53 is specific to ES cells and is not observed in other somatic cell types or adult stem cells. The dual functions of p53 to promote or repress ES cell differentiation are reminiscent of its pro- and anti-apoptotic roles in somatic cells.²⁴ In response to mild, reparable damage in somatic cells, p53 primarily triggers growth arrest, allowing DNA repaired cells to re-enter normal cell cycle; however, p53 activation after severe or irreparable DNA damage often leads to apoptosis. By analogy, the precise functional readout of p53 in ES cells under different contexts may be determined by the strength of the DNA damage stress and orchestrated by different subsets of p53 targets. This evidence illustrates the functional complexity of p53 regulation in ES cells and implicates p53 as a “decision maker” to determine whether murine ES cells undergo differentiation.

The Classic p53 Pathway as a Barrier for Somatic Reprogramming

Differentiated somatic cells can be programmed to generate induced pluripotent stem (iPS) cells that functionally resemble ES cells in their self-renewal capacity and developmental potentials. This process reflects the remarkable plasticity in development and can be triggered by exogenous expression of multiple defined transcription factors,^25-27 small molecules,^28-31 mRNAs,³² proteins³³ or even microRNAs.^34-36 iPS cells can give rise to all three germ layers and generate viable and germline-competent animals in the tetraploid complementation assay.^37,38 Despite the great promise of iPS cells in regenerative medicine, current technology frequently generates iPS cells with low efficiency and incomplete pluripotency, suggesting the existence of molecular roadblocks that suppress the reprogramming process.

In recent studies, tumor suppressors have emerged as the predominant roadblocks of somatic reprogramming. In particular, p53 is induced during somatic reprogramming and is an essential suppressor of reprogramming kinetics and efficiency.^39-44 In addition, Arf, a key regulator that activates the p53 pathway by repressing Mdm2, has been similarly characterized as a barrier to reprogramming.^42,44 The characterization of the p53 pathway as a roadblock for both tumorigenesis and reprogramming raises the possibility of mechanistic overlaps between these two seemingly distinct biological processes.^45,46

Like the multiple p53 downstream targets that collectively mediate its tumor suppressor effects, multiple downstream targets also mediate p53 suppression of reprogramming. The first p53 target identified as a suppressor of somatic reprogramming is the CDK inhibitor p21, which exhibits p53-dependent induction during reprogramming.^40,42,47 Upon p53 activation, p21 induces potent cell cycle arrest, and the resulting decrease in cell proliferation is an important barrier to somatic reprogramming.^40,48 Although, in one study, p53 knockdown and p21 knockdown generated similar increases in the stochastic reprogramming of cultured B cells,⁴⁷ careful comparison of p21^-/- and p53^-/- mouse embryonic fibroblast (MEF) reprogramming indicates a partial phenocopy of p53 by p21.^42,47 This discrepancy among these studies may reflect different experimental conditions for somatic reprogramming, different cell types used for reprogramming or, possibly, technical limitations of RNA interference in the p53 and p21 knockdown study.

The increased reprogramming efficiency of p53^-/- MEFs is also attributable to a defective apoptosis in response to DNA damage during iPS cell generation. The p53-dependent apoptosis was triggered immediately upon the introduction of the classic reprogramming factors and when pluripotency was established.^43,49 The p53 targets that mediate apoptotic response in the context of tumor suppression are well-characterized. It is likely that the same p53 targets, including Bax, Puma and Noxa, also function in the suppression of somatic reprogramming. We recently demonstrated that knocking down Bax, Puma or Noxa significantly promoted the efficiency of somatic reprogramming, partially recapitulating the effects generated by p53 deficiency (Choi YJ et al., unpublished results). The p53-mediated apoptosis during reprogramming removes cells with genetic abnormalities from the pool of potential iPS cells, thus maintaining the genomic integrity of the resulting pluripotent stem cells.⁴³ Not surprisingly, p53 deficiency increases reprogramming efficiency, yet it also compromises the functional pluripotency of the resulting iPS cells.⁴⁰

The relative contribution of proliferation and apoptosis to p53’s effects on reprogramming remains a subject of debate. In a stochastic B-cell reprogramming system, where all reprogrammed B cells eventually become iPS cells, p53 inhibits only the kinetics of somatic reprogramming, primarily due to its p21-mediated anti-proliferative effects.⁴⁷ In contrast, during MEF reprogramming, where only a small percentage of cells exhibit iPS cell-like morphology, p53 represses both the kinetics and efficiency of somatic reprogramming through mechanisms dependent and independent of cell proliferation.^49,50 Consistent with this difference, when wild-type and p53-deficient MEFs are serum starved, the p53-knockout MEFs still exhibit higher reprogramming efficiency, even though their proliferation rate is comparable to that of the wild-type MEFs.⁴⁴ These findings indicate that proliferation is not the only p53-mediated effect that suppresses MEF reprogramming, highlighting the cell type-dependent differences in p53 response during somatic reprogramming. Interestingly, at least in the context of MEF reprogramming, suppression by p53 cannot be completely explained by its effects on cell proliferation and apoptosis, suggesting that additional p53 targets mediate this effect.

p53-Regulated miR-34 miRNAs as Barriers for Somatic Reprogramming

Although the majority of characterized p53 targets are protein-coding genes, it is increasingly clear that multiple non-coding RNAs, in particular microRNAs (miRNAs), are integral components of the p53 pathway. miRNAs encode a class of small non-coding RNAs, 18–25 nucleotides in length, that regulate specific mRNA targets through post-transcriptional gene repression.^51-53 Each miRNA often recognizes multiple mRNA targets through imperfect base pairing, subsequently mediating mRNA degradation and/or translation repression.^54,55 To date, a number of miRNAs have been identified as bona fide p53 targets, modulating diverse biological processes downstream of p53 (Table 1).^56‑66 These p53-regulated miRNAs constitute a major mechanism through which activated p53 indirectly represses gene expression at the post-transcriptional level. The p53-miRNA axis, combined with the ability of p53 to directly silence gene expression by transcriptional repression, facilitates p53’s role as a potent negative regulator of gene expression. This repressive function of p53, combined with the well-characterized p53 function as a transcriptional activator, underlies many downstream biological processes during tumor suppression and somatic reprogramming.

Table 1. Direct microRNA targets of p53.

Name (family)	Examples of Targets	Function	Reference
miR-34 (miR-34a, miR-34b, miR-34c)	Ccnd1, Ccne2, Cdk4, Cdk6, E2F3, E2F5, CREB, Cdc25c, HMGA2, Ccnd3, Ccng2, Mad2L2, Mcm2, Mcm5, Plk1, c-Myc, N-Myc	Cell cycle/proliferation	Reviewed in 124 and 125
	Bcl2	Pro-apoptosis	65
	Dll1, Notch1, Notch2, Jag1	Notch signaling	65, 126–130
	SIRT1, YY1, Hdmx	p53 activity	131–134
	Nanog, Sox2, Bmi1, CD133, Olfm4, Lin28a	Stemness/pluripotency	49, 75, 78, 118
	β-catenin, Lef-1, Wnt1, Wnt3, Lrp6, Auxin2	Wnt signaling	72, 79, 128, 135
	Araf, Pik3r2, Axl, c-Met	Growth factor signaling	60, 125, 136
	Snail, Slug, Zeb1, CD44	Migration/metastasis	78, 79, 118
	Foxp1	Differentiation	137
miR-145	Oct4, Sox2, Klf4, Sox9, Elk1, Myocd, CamkIIδ	Pluripotency/lineage specific genes	76, 112, 138
	c-Myc	Proliferation	63
	EGFR, Nudt1, ERa, IRS1, Erk5, Akt, Tirap, Traf6, YES, Stat1	Signal transduction	139–142
	Srgap1, Srgap2, Add3, SSh2, MRTF-B, Actb, Fscn1	Cytoskeleton /migration	141, 143–146
miR-192, miR-194, miR-21	Rb1	Proliferation	147
	Igf1, Igf1r	IGF signaling	148
	Mdm2	p53 activity	148
	Zeb1, Zeb2	EMT/migration	61
miR-107	HIF-1β	Hypoxia	64
	Cdk6	Cell cycle control	149
	PKCε	Signal transduction	150
miR-200 (miR-200a, miR-200b, miR-200c, miR-141, miR-429)	Zeb1, Zeb2, Bmi1, Sip1	EMT	57, 151–153
	Sec23a	Protein secretion	154
	Sox2, Klf4	Pluripotency genes	153
miR-1204	n.a.	p53-mediated cell death	155

The first miRNAs identified as direct p53 targets were the miR-34 family miRNAs, including miR-34a, miR-34b and miR‑34c.^{58,60,62,65,66} The three miR-34 miRNAs are located in two separate genomic loci. The functional readout of miR-34 miRNAs, at least in overexpression studies, depends on cell type and biological contexts and comprises a broad range of biological processes downstream of p53, including cell cycle, cellular senescence, apoptosis, stem cell differentiation and mesodermal development.^{58,60,62,65-72} Consistent with miR-34 miRNAs being integral components of the p53 tumor suppressor network, genomic loss and/or epigenetic silencing of miR-34 miRNAs have been observed in a variety of human tumor types.

The mechanistic overlap between malignant transformation and somatic reprogramming has prompted efforts to ascertain the role of miR-34 miRNAs in iPS cell generation. p53 induces miR-34 miRNAs during reprogramming, and miR-34 deficiency partially recapitulates the increase in reprogramming efficiency caused by p53 deficiency. Unlike p53 loss, which enhances reprogramming at the expense of iPS cell pluripotency, genetic ablation of mir-34a promotes iPS cell generation without compromising self-renewal or differentiation.⁴⁹ The miR-34 miRNAs, particularly miR-34a, cooperate with p21 to mediate suppression of somatic reprogramming by p53.⁴⁹ Although enforced miR-34a expression leads to cell cycle arrest, senescence and apoptosis in a cell type- and context-dependent manner,^{58,60,62,65,73} miR-34a deficiency in MEFs does not have a significant proliferative advantage or apoptotic protection during somatic reprogramming.⁴⁹ These surprising findings suggest that miR-34a suppresses somatic reprogramming, largely through a mechanism independent of cell cycle control or apoptosis.

Interestingly, miR-34a deficiency causes a significant delay in iPS cell differentiation upon leukemia inhibitory factor (LIF) and feeder cell withdrawal, both in the presence and absence of RA.⁴⁹ Consistent with this observation, miR-34a specifically targets multiple key pluripotent genes, including Nanog, Sox2 and N-myc in mouse ES cells.^49,74 Derepression of these genes in miR‑34a^-/- iPS cells inhibits rapid iPS cell differentiation in response to LIF withdrawal. In addition, miR-34 represses canonical Wnt signaling by targeting β-catenin in human cancer cells and Xenopus embryos.⁷² Given the importance of Wnt signaling in maintaining pluripotent stem cells’ capacity to self-renew, the p53-miR-34-Wnt pathway also likely regulates somatic reprogramming. Finally, miR-34a also targets Lin28a during somatic reprogramming, which enhances reprogramming in both mouse and human fibroblasts.^47,75 Taken together, this evidence suggests that during reprogramming, post-transcriptional derepression of pluripotency genes resulting from miR-34a deficiency promotes and accelerates establishment of the endogenous regulatory circuitry for pluripotency.

It is worth noting that miR-34-mediated gene silencing is not the only mechanism for repressing pluripotency genes downstream of p53. p53 also downregulates pluripotency genes through additional direct and indirect mechanisms. For example, p53 directly represses Nanog expression by recruiting mSin3a to the Nanog promoter,²⁰ and p53 regulates additional miRNAs, such as miR-145, to repress key pluripotency genes (see below). As a global gene regulator, p53 activates and suppresses gene expression, and both mechanisms cooperatively inhibit somatic reprogramming.

The p53-miRNA Network Regulates Stem Cell Pluripotency and Somatic Reprogramming

In addition to miR-34 miRNAs, a number of other miRNAs have been identified as bona fide p53 targets, including miR‑192, miR‑194, miR-215, miR-107, miR-200 and miR-145 (Table 1). Several of these additional p53-regulated miRNAs similarly function at the crossroads of tumor suppression and somatic reprogramming. miR-145, for instance, is activated by p53 through both transcriptional activation and post-transcriptional maturation.^13,76 miR-145 is expressed at low levels in embryonic stem cells, largely due to direct transcriptional repression by Oct4. Differentiation induces miR-145 in human ES cells, which then targets multiple pluripotency genes Oct4, Sox2 and Klf4 to promote differentiation.⁷⁶ Consistent with its role in promoting differentiation, a recent study demonstrated that miR-145 is a downstream effector of p53, negatively regulating the reprogramming process. These findings have revealed a novel cellular and molecular mechanisms underlying the p53 suppression of somatic reprogramming.⁷⁵

miR-200c is another p53 transcriptional target implicated in somatic reprogramming. The miR-200 family of miR NAs, including miR-200a, miR-200b, miR-200c, miR‑141 and miR-429, were initially characterized as inhibitors of epithelial-to-mesenchymal transition (EMT) that functioned by repressing Zeb1, Zeb2 and Bmi1.^57,61 Despite the importance of EMT in development, physiology and tumor invasion, EMT and its reverse process, MET (mesenchymal-to-epithelial transition), were characterized only recently in pluripotent stem cell differentiation and somatic reprogramming, respectively. MET is the initial step of MEF reprogramming upon introduction of reprogramming factors, which correlates with induction of miR-200 miRNAs in a BMP-dependent manner.⁷⁷ Consistently, enforced miR-200 expression enhances MET and facilitates MEF reprogramming. In fact, miR-200 miRNAs promote reprogramming so potently that miR-200c, miR-302 and miR-367 miR NAs alone are sufficient to generate iPS cells in the absence of any protein-coding reprogramming factors.³⁶ Given the ability of p53 to induce miR-200c in multiple cell types, these findings are at odds with the suppressive role of p53 during iPS cell generation as well as miR-200c-mediated repression of stemness by p53 in breast cancer cells.⁵⁷ These observations raise several questions about the role of the p53-miR-200c axis in iPS cell generation: Is miR-200c induced in a p53-dependent manner during somatic reprogramming, as it is in cancer cells and MEFs? Are the effects of MET on stemness and somatic reprogramming cell-type dependent? Finally, does p53 play dual roles at different stages of MEF reprogramming, with the overall effect as a suppressor to reprogramming? Since the ectopic miR-34 expression induces MET by targeting Snail, Slug and Zeb1, could the p53-miR-34 axis also play dual roles at different stages of reprogramming?^78,79 The answers to these questions will further dissect the p53 pathway in the context of somatic reprogramming to reveal its complex functional impacts.

miRNAs are integral components of the p53 pathway, not only acting as direct p53 targets to mediate multiple biological effects downstream, but also targeting key components of the p53 (or p53 family) network.^80-82 For example, the miR-290 miRNA cluster contains seven miRNAs that constitute 70% of total miRNAs expressed in ESCs.⁸³ Among the targets of miR-290 cluster are p21 and Ei24, canonical p53 targets and Lats2, a positive modulator of p53 activity.^84-86 In the ES cell context, high-level expression of the miR-290 cluster dampens activity of the p53 pathway, expediting the G₁/S transition in cell cycle. The miR‑290 cluster contains components that share seed sequence homology with those of the mir-17–92, mir-106a-363 and mir-106b-25 miRNA clusters. This family of homologous miRNAs collectively targets the canonical p53 target p21, thus promoting somatic reprogramming efficiency by accelerating cell proliferation.⁸⁰ Moreover, the effects of these miRNAs on somatic reprogramming are also achieved by promoting MET through targeting the components of the TGFβ pathway.⁸⁰

A number of miRNAs also modulate the activity of the p53 pathway and, therefore, may regulate the biological properties of pluripotent stem cells and somatic reprogramming.⁸¹ For example, the transcriptomes of human pluripotent stem cells, including ES cells and iPS cells, can be classified into two distinct states, irrespective of cell type of origin.⁸⁷ The major difference between these two pluripotency states is the activity of the p53 network. Although the functional significance of the two pluripotent states remains unclear, these two states can be defined by the expression level of two miRNAs that directly target p53, miR-92 and miR-141.⁸⁷ More significantly, overexpression of these miRNAs is sufficient to alter pluripotency status, at least at the transcriptome level.⁸⁷ The effect of miR‑21 and miR-29a on reprogramming is another example where p53 regulatory miRNAs control the establishment of pluripotency. miR-21 and miR-29a modulate p53 level by repressing negative regulators of the p53 pathway, such as YY1, p85 and CDC42.^81,88,89 Consistent with p53 as a major roadblock of somatic reprogramming, inhibition of miR-21 and miR-29a enhances the efficiency of iPS cell generation. Additionally, a number of miRNAs, including miR‑125, miR-380-5p and miR-33, are regulators of p53 activity in different contexts.^90-92 Whether any of these miRNAs play a role in somatic reprogramming remains to be determined.

Taken together, p53 presents a potent roadblock to somatic reprogramming induced by the classic ES cell-specific transcriptional factors (Figs. 1 and 2). This effect is mediated through at least three cellular and molecular mechanisms: p53 induces p21 to repress cell proliferation; p53 activates Bax, Puma and Noxa to trigger apoptosis in response to DNA damage during iPS cell generation; and p53 directly or indirectly downregulates multiple key pluripotency genes to inhibit the establishment of pluripotency (Fig. 2). The miRNAs within the p53 network are essential regulatory players in all three mechanisms, either by mediating the p53 downstream effects or by modulating p53 activity.

Figure 1. The p53-miRNA network in somatic cell reprogramming. p53 regulates reprogramming process by activating different miRNAs modulating different processes, such as stemness or EMT. miRNAs can also affect reprogramming by targeting p53 itself, by targeting p53 modulators, or by targeting p53 downstream effectors of reprogramming. Dashed line indicates the regulations not yet identified in the ES or iPS cell context.

Figure 2. p53 regulates reprogramming and transformation through shared mechanisms. p53 regulates reprogramming and transformation by controlling proliferation and cell survival. In addition, p53 can control reprogramming by repressing pluripotency genes at both transcriptional and post-transcriptional (miRNA) levels. It remains to identify whether the p53-miR-34-pluripotent gene axis or the reprogramming process also plays roles in tumorigenesis.

The p53 Network in Adult Stem Cells

Not only does the p53 pathway play an important role in pluripotent stem cells, it also functions to regulate the self-renewal, cell proliferation and genomic integrity of adult stem cells.^93-95 In adult mice, p53 deficiency consistently increases the proliferation and self-renewal of neural stem and progenitor cells in the subventricular zone (SVZ) of the brain.^96-98 p53 also regulates the differentiation of neural progenitor cells and oligodendrocyte precursor cells in vitro.^99-101 These findings are consistent with the exencephaly phenotype observed in p53-knockout females,^102,103 which is attributed to the failure of neural tube closure due to outgrowth of neural tissues.¹⁰³ Several p53 protein-coding targets, such as Doux1 and Douxa1, regulate neuronal differentiation in neural stem cells.¹⁰⁴ But the ability of the p53-miR-34 pathway to repress Sox2 and the Wnt pathway suggests new potential mechanisms for the maintenance or differentiation of adult neural stem cells in SVZs.

The p53 activity also inhibits the proliferation, self-renewal and repopulating potential of hematopoietic stem cells (HSCs) in mouse models. Most HSCs are quiescent, but some HSCs can differentiate into a variety of progenitor cells, which replenish the hematopoietic system.¹⁰⁵ p53 plays a crucial role in maintaining the quiescent state of HSCs and controlling the self-renewal of HSCs through a p21-independent mechanism.¹⁰⁶ In addition, aged p53^+/- mice have significantly more proliferating HSCs compared with their wild-type counterparts, whereas HSCs from mice carrying one hyperactive p53 allele (p53^+/m) have compromised self-renewal and proliferation activity, which may account for the early aging phenotype.¹⁰⁷ Similar to its role in somatic cells, p53 protects HSCs from DNA damage and mutation by controlling the levels of reactive oxygen species, the major cause of DNA damage and genomic instability in these cells.¹⁰⁸ Interestingly, p53 regulates the homeostasis of hematopoietic stem and progenitor cells (HSPCs) through a cell-cell competition mechanism.¹⁰⁹ In the absence of an apparent DNA damage response, HSPCs with low p53 levels out-compete the ones with high p53 levels.¹⁰⁹ Induction of senescence genes in HSPCs with high p53 level mediates this selective expansion.¹⁰⁹ Taken together, these results suggest that p53 may play important roles in HSCs or HSPCs. Moreover, it has been demonstrated that miR-33 controls self-renewal of mouse HSCs through targeting p53,¹¹⁰ further supporting the involvement of p53-miRNA network in modulating HSC function. It remains to be determined if miRNA components of the p53 network regulate the proliferation, self-renewal, differentiation and genomic integrity of HSCs.

Similar to other adult stem cells, p53 deficiency results in genomic instability in mesenchymal stem cells (MSCs).¹¹¹ However, in contrast to its differentiation-promoting function in neural stem cells and mammary gland stem cells (MGSCs), p53 seems to suppress the differentiation of MSCs, since p53-deficiency in MSCs reduces the proliferation rate and accelerates differentiation of osteocytes, myofiblasts or adipocytes.^94,111 Mechanistic studies indicate the ability of p53 to repress important transcription factors that induce differentiation in MSCs, including Myocd, PPARγ and osterix.⁹⁴ Interestingly, miR-145, a p53-regulated miRNA described before, also regulates smooth muscle cell differentiation and chondrogenic differentiation.¹¹² Therefore, it is possible that the miRNAs contribute to the regulation of MSC differentiation by p53.

p53 at the Crossroads between Stem Cells and Tumor Suppression

Despite decades of p53 research, we are only starting to understand the molecular mechanisms that constitute the crosstalk between tumor suppression and stem cell biology downstream of p53. Given the considerable mechanistic overlap between somatic cell reprogramming and malignant transformation, it is intriguing to compare the generation of iPS cells in reprogramming and cancer initiating cells during tumorigenesis. Significantly, recent studies have suggested potential correlation between the status of p53 and the stemness of the cancer cells. In the context of breast cancer, mutant p53 is associated with a stem cell transcriptome signature, and loss of p53 in mammary epithelial cells activates EMT, accompanied by an increase in mammary stem cell population. In particular, normal and cancerous MGSCs differ in their self-renewing division, with normal stem cells having more asymmetric division and cancer stem cells having more symmetric cell division.¹¹³ p53 controls the switch between these two types of self-renewal, as normal MGSCs with mutant p53 resemble cancer stem cells and divide symmetrically.¹¹³ Such symmetric division ultimately increases total cell number and contributes to tumor growth.¹¹³

Besides controlling the pattern of cell division, p53 also controls the self-renewal of tumor initiating cells. The leukemia stem cells (LSCs) are a subpopulation of cancer cells that possess stem cell-like properties (i.e., they are quiescent but retain the ability to differentiate). Due to their quiescence, LSCs are resistant to chemotherapy and are thought to be responsible for the relapse of leukemia after treatment.¹¹⁴ As with its role in controlling quiescence and self-renewal in HSCs, p53 also controls the self-renewal of LSCs in mouse models.^115,116 In an adoptive transfer AML model, overexpressing a short isoform of MEL1 in combination with p53 deficiency enhances the self-renewal of LSCs.¹¹⁵ In another AML model driven by Kras^G12D, p53 limits the aberrant self-renewal of myeloid progenitor cells caused by oncogenic K-Ras.¹¹⁶ Although the exact molecular mechanisms underlying this regulation still remain largely unknown, these results suggest that p53 suppresses the self-renewal, proliferation or immortalization of LSCs.¹¹⁶

The dual functions of the p53 pathway in tumor suppression and in stem cell biology reflect the considerable mechanistic overlap between malignant transformation and stem cell reprogramming. Accumulating evidence suggests that miRNA components within the p53 network connect these two arms of p53 function. Although direct evidence of in vivo reprogramming is still lacking, emerging evidence suggests that the p53-miRNA network may inhibit formation of tumor-initiating cells generated by repressing dedifferentiation. As key p53 targets, miR-34 miRNAs downregulate multiple stem cell-specific genes that are enriched in putative cancer-initiating cell populations and poorly differentiated human tumors.¹¹⁷ Two recent studies identified miR-34a as a key suppressor of cancer stem cells in prostate tumors and gliomas, acting, at least in part, by downregulating CD44 (in prostate tumor) or by downregulating c-Met and Notch pathway (in glioma).^118,119 Conceivably, the ability of miR-34 miRNAs to repress Nanog, Sox2 and N-myc may also contribute to the repression of stemness in the context of tumorigenesis. Two additional p53 miRNA targets, miR-145, a repressesor of pluripotency genes, and miR-200c, a repressesor of EMT and EMT-associated stem cell properties, could also limit the stemness of cancer cells. Overall, p53-regulated miRNAs constitute an essential mechanism of tumor suppression by reducing the stemness of tumor cells and repressing cellular dedifferentiation.

Besides miRNAs, other ncRNAs, particularly long-intergenic non-coding RNAs (lincRNAs), are also integral components in the p53 pathway.^120,121 LincRNAs are evolutionarily conserved, long non-coding RNAs that regulate diverse biological processes by modulating the epigenetic status of chromatin.¹²⁰ Two p53-regulated lincRNAs, TUG1 and lincRNA-p21, mediate acute p53 response.^121,122 Moreover, dozens of lincRNAs play functional roles in self-renewal or differentiation of murine ES cells.¹²³ It remains to be determined whether the lincRNAs, together with p53-regulated protein-coding genes and miRNAs, constitute a network of effectors that mediate p53 functions at the crossroads between tumor suppression and stem cell biology. There is no doubt that elucidating the functions of non-coding RNAs in the p53 network will bring fundamental insights into the diverse biological functions of p53.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.