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p53, stem cell biology and childhood blastomas

Oh, Lixiana; Hafsi, Hindb; Hainaut, Pierreb; Ariffin, Hanya

doi: 10.1097/CCO.0000000000000504
CANCER BIOLOGY: Edited by Pierre Hainaut
Free
SDC

Purpose of review Childhood blastomas, unlike adult cancers, originate from developing organs in which molecular and cellular features exhibit differentiation arrest and embryonic characteristics. Conventional cancer therapies, which rely on the generalized cytotoxic effect on rapidly dividing cells, may damage delicate organs in young children, leading to multiple late effects. Deep understanding of the biology of embryonal cancers is crucial in reshaping the cancer treatment paradigm for children.

Recent findings p53 plays a major physiological role in embryonic development, by controlling cell proliferation, differentiation and responses to cellular stress. Tumor suppressor function of p53 is commonly lost in adult cancers through genetic alterations. However, both somatic and germline p53 mutations are rare in childhood blastomas, suggesting that in these cancers, p53 may be inactivated through other mechanisms than mutation. In this review, we summarize current knowledge about p53 pathway inactivation in childhood blastomas (specifically neuroblastoma, retinoblastoma and Wilms’ tumor) through various upstream mechanisms. Laboratory evidence and clinical trials of targeted therapies specific to exploiting p53 upstream regulators are discussed.

Summary Despite the low rate of inherent TP53 mutations, p53 pathway inactivation is a common denominator in childhood blastomas. Exploiting p53 and its regulators is likely to translate into more effective targeted therapies with minimal late effects for children. (see Video Abstract, Supplemental Digital Content 1, http://links.lww.com/COON/A23).

aDepartment of Paediatrics, University of Malaya, Kuala Lumpur, Malaysia

bInstitute of Advanced Biosciences, University of Grenoble-Alpes, La Tronche, France

Correspondence to Hany Ariffin, MBBS, PhD, Department of Paediatrics, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Tel: +60 3 7949 2416; e-mail: hany@um.edu.my

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (www.co-oncology.com).

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INTRODUCTION

Blastomas are a heterogeneous group of primitive neoplasms, more common in children than in adults, where tumor cells are composed of immature undifferentiated cells resembling those of the blastoma or primordium of the organ in which the tumor arises. Examples of childhood blastoma include neuroblastoma, nephroblastoma (Wilms’ tumor), retinoblastoma, hepatoblastoma and several very rare forms, namely pleuropulmonary blastoma and pancreatoblastoma [1]. These cancers, sometimes referred to as ‘embryonal cancers,’ are predominantly located in developing tissues of children younger than 4 years [2]. They are postulated to arise from embryonic cells that fail to undergo normal maturation during development [3]. Collectively, the age-standardized incidence rates of embryonal cancers in the United States and Europe are estimated to about 5.5 per million (2.4 neuroblastoma, 1.9 Wilms’ tumor, 0.8 retinoblastoma and less than 0.5 for other types). Survival rates are generally high (greater than 90% for Wilms’ tumor and retinoblastoma, 71% for hepatoblastoma, 68% for neuroblastoma) with the best outcome seen in children aged 0–14 years [2]. However, certain subtypes have much lower survival rates despite intensive treatment. For example, high-risk neuroblastoma is highly lethal, with a 5-year overall survival of only 40–50% [4].

A recent analysis of the landscape of genomic alterations across 24 types of childhood cancers has revealed marked differences in terms of mutation frequency and significantly mutated genes (SMGs) in comparison with most adult cancers [5▪▪]. This analysis has shown that mutation frequencies in childhood cancers ranged between 0.02 and 0.49 mutations per Mb, on average 14 times lower than in adult cancers (0.13 mutations per Mb in childhood cancers versus 1.8 in adults). Among childhood cancers, embryonal cancers are those with the lowest mutation rates, ranging between 0.06 (retinoblastoma) and 0.23 (neuroblastoma) mutations per Mb (average: 0.15, versus 1.1 for all other childhood cancers, thus seven times lower). With few exceptions, mutations in SMGs are rare. Although causative genetic alterations underlying the initiation of blastomas have been identified, including RB1 mutation or loss in retinoblastoma, WT1 mutation or deletion in Wilms’ tumor and MYCN amplification, chromosome 1p loss or gain of chromosome 17q in neuroblastoma, it is becoming increasingly evident that other genetic, epigenetic, or protein activation events are also necessary for development of these tumors [5▪▪,6▪,7,8,9].

TP53, the most frequently altered gene in all adult cancers (on average >50%), is mutated in 0% of hepatoblastoma and retinoblastoma, in 1.8% of neuroblastoma and in 7.8% of Wilms’ tumor, much lower than the 12% average observed across 20 other childhood cancer types. These mutations are mostly associated with tumors that relapse after treatment [5▪▪]. This exceptionally low mutation frequency suggests that alternative mechanisms are at work to circumvent the tumor suppressive activities of the p53 protein in embryonal cancers, thus making inactivation by mutation not necessary for these cancers to develop and progress. In this review, we summarize current knowledge and hypotheses regarding alternative mechanisms of p53 inactivation that may operate in neuroblastoma, Wilms’ tumor and retinoblastoma, and we discuss the therapeutic potential of using targeted small molecules to restore the activity of wild-type p53.

Box 1

Box 1

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P53, STEM CELL BIOLOGY

The p53 protein is an inducible transcription factor, which is expressed at only low levels in most normal cells but undergo rapid nuclear accumulation and activation in response to multiple forms of stress, including chemical or radiation-induced DNA-damage, hypoxia, nutrient deprivation, disruption of cell–cell or cell–matrix contacts [10,11]. Following activation, p53 operates as a transcriptional activator for a broad range of genes involved in cell cycle arrest, apoptosis, senescence, differentiation, metabolism, immune response, and DNA repair. Activated p53 also contributes to many of these stress responses through nontranscriptional mechanisms, such as binding to other proteins or relocating in critical cell compartments, such as the mitochondria [10,12]. Activation of p53 involves massive posttranslational modification and results in protein stabilization by escape from degradation by the proteasome. The kinetics and extent of p53 activation is dependent upon cell and tissue context, in addition to the nature and intensity of inducing signals, thus defining an extremely diverse repertoire of growth suppressive responses. Mutations in TP53, by disrupting its capacity to act as a transcription factor, collectively switches off most of these responses, thus removing an integrated brake for cancer development and progression.

p53 is ubiquitous and is present in cells at all stages of their life cycle, from stem cells to postmitotic, terminally differentiated and senescent cells. There is evidence that the status of p53 as an inducible factor varies from one stage to another, thus defining a cell lifetime trajectory of p53 activity that regulates cell fate [13▪]. Specifically, the p53 protein is expressed in stem cells, in which it plays both active and passive roles. In stem cells, the transcriptional activities of p53 is at least partially turned-off, with attenuated capacity to induce cell cycle arrest and apoptosis, whereas regulatory roles in cell bioenergetic metabolism appear to be preserved [13▪]. Stabilization of p53 in stem cells promotes the commitment to differentiation and the formation of progenitor cells. In the absence of p53, stem cells accumulate genetic damage caused by errors in replication and DNA repair, thus leading to enhanced proliferation of cells with potentially oncogenic DNA damage. Maintenance of p53 in stem cells, thus contributes to their genetic stability and long-term viability. In p53-competent cells, the addition of the four so-called Yamanaka transcription factors c-Myc, Klf-4, Oct4, and Sox2 to fibroblastic cells in culture results in a small percentage of cells (0.1–1%) undergoing reprogramming to form induced pluripotent stem cells (iPS) [14,15]. In cells lacking p53, the reprogramming of fibroblasts to iPS cells can occur with only Sox2 and Oct4, at an accelerated rate and in a higher percentage of the cells (10–80%) [15–19]. However, the resulting iPS appear to be more genetically unstable, thus showing that p53 is acting as both a rate-limiting factor and a promoter of fidelity in the maintenance of stem cells.

The molecular mechanisms that control p53 activity in stem cells are only partially understood. Critical mechanisms include deacetylation of p53 by SIRT1 [20,21], aurora kinase A-mediated phosphorylation [22], or expression of p53 isoforms such as Delta40p53, which lacks the N-terminal transactivation domain, engage in the formation of tetramers with full-length p53 and attenuates its transcriptional activity [23].

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ALTERATIONS OF THE P53 PATHWAY IN BLASTOMAS

Given that blastomas are considered as deriving from abnormal stem cells, it is tempting to speculate that their low frequency of inactivating mutations in TP53 is because of their retention of regulatory mechanisms that attenuate p53 function, thus making mutation dispensable for cancer development (Table 1 and Fig. 1). Indeed, blastomas express wild-type p53 in more than 90% of the cases and are rarely detected in individuals with Li-Fraumeni syndrome, a familial cancer predisposition associated with germline TP53 mutation [24–26]. Mechanisms that may contribute to down-regulate p53 in blastoma include, among others, upregulation of Mdm2/Mdmx, loss of upstream activators, such as p16INK4a/p14ARF, inactivation by microRNA targeting the p53 pathway, specific patterns of inactivating posttranslational modifications and over-expression of dominant-negative p53 isoforms.

Table 1

Table 1

FIGURE 1

FIGURE 1

During embryonic development, p53 is tightly regulated by its critical Mdm2/Mdmx negative regulatory feedback system. Both Mdm2 and Mdmx proteins interact with p53 at its N-terminus and regulate protein stability and transcriptional activity [27]. Thus, increased expression or activity of these two nonredundant p53 inhibitors could attenuate p53-mediated tumor suppression. Inactivation of p53 pathway through MDM2 and/or MDMX amplification has been observed in both retinoblastoma and neuroblastoma [28–30]. Mdm2 and Mdmx protein expression or activity in blastomas can also be modulated by single nucleotide polymorphism SNP309T>G [31–34], by overexpression of MYCN [35–39] and by alternative splicing of MDMX mRNA [40]. Taken together, these data support the indication of targeting the interaction of Mdm2/Mdmx with p53 as a possible therapeutic approach.

Mdm2/p53 binding is antagonized by the tumor suppressor p14ARF protein, encoded by the alternative transcript of CDKN2A locus [41,42]. Loss of p14ARF function would enhance the inhibitory effect of Hdm2 towards p53, thus abrogating p53-mediated tumor suppression. Indeed, p14ARF inactivation either by deletion or promoter hypermethylation has been reported in neuroblastoma (29–35%) [28,43], Wilms’ tumor (∼5%) [44] and retinoblastoma (∼15%) [45]. Levels of p14ARF in childhood blastomas may also be modulated by its upstream regulators. In both primary tumors and xenograft models, overexpression of Bmi-1 [46,47], enhanced level of Twist1 [48,49] or reduced expression of Chd5 [50,51] have been reported in Wilms’ tumor and neuroblastoma. In neuroblastoma, one copy of CHD5 is frequently lost, whereas the remaining allele is seldom altered but frequently silenced by promoter methylation [51].

Negative regulation of p53 by miRNAs can occur through direct binding to p53 mRNA (e.g. miR-504, miR-125b, miR-380–5p) or indirectly through repression of p14ARF (by miR-24). High levels of these miRNAs have been reported in embryonal cancers, particularly retinoblastoma [52,53] and neuroblastoma [54,55]. Some tumor suppressive miRNAs, which upregulate p53 activity through direct suppression of MDMX (by Let-7 [56,57], miR-191 [40]), MDM2 (by miR-192, miR-194, miR-215 [58,59]), SIRT1 (by miR-34a [60–63]) or cyclin G1 (by miR-122 [64,65]), were reported to be weakly expressed in blastomas. Dysregulation of miRNA processor genes, such as DICER and DROSHA was demonstrated in high-risk neuroblastoma [66] and primary Wilms’ tumor [67], further highlighting the significance of miRNA disruption in activating p53 in embryonal cancers.

The activity of p53 protein can also be regulated through TP53 gene polymorphisms and other posttranslational regulators. p53-72P variant (rs1042522; G>C), which is considered as less effective in inducing apoptosis than p53-72R is associated with development or disease progression of blastomas [68–72]. In neuroblastoma, another rare germline TP53 variant at the polyadenylation signal (rs78378222 A>C), which impairs the 3’-end processing of TP53 mRNA, was identified to associate with susceptibility to neuroblastoma [73]. Studies in both neuroblastoma and Wilms’ tumor have demonstrated that p53 can be inactivated posttranslationally through reduced acetylation (in relation to frequent loss or mutation of WTX [74,75]), Ser215/315-phosphorylation (by upregulation of aurora-kinase A [76–80]) and lysine methylation (by high levels of SETD8 [81]). A recent siRNA and chemical screen has identified the lysine methyltransferase SEDT8 as a therapeutic target for p53 activation in high-risk neuroblastoma [82▪▪]. Other studies have suggested that overexpression of Twist1 transcription factor and Bmi-1 polycomb complex protein may also inactivate p53 via direct binding, enhanced ubiquitination and degradation as well as inhibiting posttranslational mechanisms [83–86].

The activity of p53 can also be fine-tuned by several p53 isoforms, expressed from the TP53 gene through alternative promoter or codon initiation usage, alternative splicing, or combinations thereof [23]. N-terminal p53 isoforms (Delta40, Delta133 and Delta160) are functionally impaired by truncation of regions of the protein containing the transactivation domain and parts of the DNA-binding domain. There is evidence that Delta40p53 (lacking the first 39 residues of p53) may function as an inhibitor of p53 transactivation and that decreased expression of this isoform is responsible for the phenotypic switch from pluripotency to differentiation in ESCs [87]. Maier et al. [88] reported that homozygote Delta40p53 mice (p53ΔNp53/ΔNp53) were prone to develop cancer, at the similar rate of p53 null (p53-/-) mice. Expression of Delta122p53, the mouse counterpart of human Delta133p53, as a transgene in the mouse confers a marked proliferative advantage on cells and reduced apoptosis, associated with a spectrum of cancers, which is both different and more severe than in p53-null mice [89]. Several studies reported an elevated expression of N-terminal p53 isoforms (Delta40p53 or Delta133p53) in a variety of adult tumors [90–94]. To our knowledge, p53 isoforms have not been systematically studied in embryonal cancers; however, evidence of splicing dysregulation and alternative promoter usage in these cancers may support our hypothesis. The dysregulated activity of splicing kinases including SRPK1 and AURKA observed in Wilms’ tumor [95] and neuroblastoma [96,97], respectively, may manipulate the balance between full-length and oncogenic DeltaNp53 in these embryonal cancers. Related to this, the TP53 SNP rs35850753 (A>G), which maps to 5′-UTR of Delta133p53 and postulated to drive TP53 alternative transcript expression, has been found to be robustly associated with neuroblastoma predisposition [73]. These findings support continued investigation into the role of p53 isoforms in term of expression, activity and regulation in embryonal cancers.

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THERAPEUTIC STRATEGIES IN CHILDHOOD BLASTOMAS

Past decades have seen dramatic improvements in survival rates for children with blastomas. However, given the long-term toxic effects of genotoxic drugs, there remains a strong clinical need to explore new therapeutic options, which are more efficacious yet associated with minimal late effects. Therefore, re-activation of p53 via targeted, nongenotoxic approaches is an attractive therapeutic strategy.

Pharmacological inactivation of Mdm2 with nutlin-3 and other Mdm2-p53 antagonists (RG7112, MK-8242, HDM201) has been evaluated in mouse xenograft of various blastomas and phase I clinical trial of neuroblastoma (NCT02780128) (Table 2). Inhibition of the Mdm2–p53 interaction in these tumors resulted in activation of p53 pathway, and consequently, induction of apoptosis, differentiation, inhibition of tumor growth and metastasis, as well as sensitization of tumor cells to cytotoxic and other molecular-targeted therapies [24,98,99▪,100]. It is worth mentioning that as shown in vitro, neuroblastoma cells may develop resistance by inducing de novo p53 mutations or overexpressing Mdmx (not targeted by Mdm2–p53 antagonists) upon continuous exposure to Nutlin-3. Thus, MDM2-p53 antagonists may be more effective when used in combination with other agents, such as Mdmx inhibitors or p53 activators in these embryonal cancers [101]. However, the toxicity profile of the combination partner is a critical determinant of the success of such an approach clinically. Indeed, the main limitation to the use of current Mdm2 antagonist is the high incidence of hematological toxicities in clinical trials, suggesting that therapies with overlapping side effect profiles would not be suitable as combination partners [102▪].

Table 2

Table 2

Another promising approach to restore p53 expression (as well as the expression of other silenced tumor suppressor genes) in blastoma is the use of epigenetic modifiers, such as genistein to decrease hypermethylation levels of tumor suppressor genes, such as CHD5 and TP53. Preclinical studies in mice carrying neuroblastoma xenografts showed that genistein could enhance the expression of CHD5 as well as p53, possibly contributing to inhibition of neuroblastoma growth in vivo and tumor microvessel formation [103]. This approach has not yet been tested in clinical studies.

To correct abnormal miRNA expression, miRNA modulation by replacing/forcing expression of suppressor miRNAs or inhibiting oncogenic miRNAs using antagomirs has been tested in blastomas. Suppression of oncogenic miR-380-5p and miR-125b in in-vivo models of mouse neuroblastoma and zebrafish brain resulted in the increase of p53 protein and subsequent apoptosis [54,55]. Conversely, transfection with tumor-suppressive miRNAs (Let-7 miRNA mimics or miR-34a) resulted in cellular differentiation and diminished tumor growth as a result of apoptosis and DNA synthesis suppression in both neuroblastoma and retinoblastoma models [62,104,105]. However, despite promising results in preclinical models, miRNA-mediated therapies have not entered mainstream cancer therapy and still require further refinement to minimize off-target effects [106].

High expression of AURKA has shown oncogenic properties in human cancers, including neuroblastoma. Cell culture and xenograft experiments have indicated that treating neuroblastoma by depleting AURKA using shRNA or inhibitors (CCT137690 and MLN8237) leads to significant tumor growth inhibition [107–109]. Some AURKA inhibitors including MK-5108 and MLN8237 have undergone Phase I and II clinical trials (NCT00543387, NCT02444884, NCT01154816) in patients with advanced/refractory and recurrent neuroblastoma as a single agent or in combination with existing chemotherapies (Table 2). It is worth noting that concomitant inhibition of Mdm2-p53 and Aurora kinase acts synergistically to induce p53-dependent apoptosis in acute myelogenous leukemia, and may be efficacious in neuroblastoma as well [110].

The finding of high p53K382me1 levels in neuroblastoma, especially in MYCN-wild type neuroblastoma supports the idea of reactivating p53 by posttranslational mechanisms. The Thiele group [81,82▪▪] was the first to demonstrate that genetic or pharmacologic (UNC0379) inhibition of SETD8 both led to a decrease in p53K382me1 levels and impair the growth of neuroblastoma xenograft tumors through rescuing the pro-apoptotic and cell cycle-arrest functions of p53.

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CONCLUSION

Low somatic TP53 mutation rates in embryonal cancers has led to the convention that p53 dysfunction is not a significant event in the development of these tumors. However, with further understanding of the p53 pathway and its regulation, p53 inactivation via multiple upstream mechanisms is evident in these tumors. As the majority of embryonal childhood cancers have wild-type p53 with intact downstream p53-signalling pathways, re-activation of p53 via targeted, nongenotoxic approaches may force cellular differentiation and result in tumor regression or a less malignant phenotype. These p53-based strategies may be promising, and less noxious in managing childhood tumors where arrested or aberrant organ development is the underlying anomaly.

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Acknowledgements

We thank all members of Paediatric Oncology Research Lab in University Malaya for their help in the making of the video abstract.

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Financial support and sponsorship

p53 research in Paediatric Oncology Research Laboratory, is supported by the University Malaya Research Grant (UMRG) (RP049B-17HTM) and by INCa Grant PLBIO16–271 ‘p53 Metabolism’ at IAB Grenoble, France.

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Conflicts of interest

There are no conflicts of interest.

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REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest
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Keywords:

childhood blastomas; differentiation; p53; stem cells; targeted therapy

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