p53 and its mutants on the slippery road from stemness to carcinogenesis

Carcinogenesis, Apr 2017

Normal development, tissue homeostasis and regeneration following injury rely on the proper functions of wide repertoire of stem cells (SCs) persisting during embryonic period and throughout the adult life. Therefore, SCs employ robust mechanisms to preserve their genomic integrity and avoid heritage of mutations to their daughter cells. Importantly, propagation of SCs with faulty DNA as well as dedifferentiation of genomically altered somatic cells may result in derivation of cancer SCs, which are considered to be the driving force of the tumorigenic process. Multiple experimental evidence suggest that p53, the central tumor suppressor gene, plays a critical regulatory role in determination of SCs destiny, thereby eliminating damaged SCs from the general SC population. Notably, mutant p53 proteins do not only lose the tumor suppressive function, but rather gain new oncogenic function that markedly promotes various aspects of carcinogenesis. In this review, we elaborate on the role of wild type and mutant p53 proteins in the various SCs types that appear under homeostatic conditions as well as in cancer. It is plausible that the growing understanding of the mechanisms underlying cancer SC phenotype and p53 malfunction will allow future optimization of cancer therapeutics in the context of precision medicine.

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p53 and its mutants on the slippery road from stemness to carcinogenesis

Carcinogenesis p53 and its mutants on the slippery road from stemness to carcinogenesis Alina Molchadsky 0 Varda Rotte 0 0 Department of Molecular Cell Biology, Weizmann Institute of Science , Rehovot 76100 , Israel Normal development, tissue homeostasis and regeneration following injury rely on the proper functions of wide repertoire of stem cells (SCs) persisting during embryonic period and throughout the adult life. Therefore, SCs employ robust mechanisms to preserve their genomic integrity and avoid heritage of mutations to their daughter cells. Importantly, propagation of SCs with faulty DNA as well as dedifferentiation of genomically altered somatic cells may result in derivation of cancer SCs, which are considered to be the driving force of the tumorigenic process. Multiple experimental evidence suggest that p53, the central tumor suppressor gene, plays a critical regulatory role in determination of SCs destiny, thereby eliminating damaged SCs from the general SC population. Notably, mutant p53 proteins do not only lose the tumor suppressive function, but rather gain new oncogenic function that markedly promotes various aspects of carcinogenesis. In this review, we elaborate on the role of wild type and mutant p53 proteins in the various SCs types that appear under homeostatic conditions as well as in cancer. It is plausible that the growing understanding of the mechanisms underlying cancer SC phenotype and p53 malfunction will allow future optimization of cancer therapeutics in the context of precision medicine. - Introduction The processes underlying normal development, tissue homeo- germ layer. Whereas, oligopotent SCs are able to give rise to a stasis and regeneration upon injury include replication of exis-t more limited subset of cell lineages than the multipotent ones. ing cells, expansion and differentiation of stem and progenitor Finally, unipotent SCs are able to produce only one mature cells, as well as trans-differentiation or dedifferentiation of cell type. Therefore, oligopotent and unipotent SCs are termed cells from one cell type to another. The hallmark character-is progenitor cells (3,4). Importantly, preservation of genomic tics of stem cells (SCs) are self-renewal, the ability to proliferate fidelity in SCs is of key importance for normal development indefinitely throughout life and the capacity to undergo d-if and regeneration. On the contrary, lack of SCs integrity may ferentiation into various cell types, upon specific signals1,(2). result in developmental abnormalities and the rise of cancer SCs persisting throughout both embryogenesis and postnatal stem cells (CSCs) that greatly contribute to various aspects of period can be classified according to their distinct different-ia tumorigenesis. tion potency. Totipotent SCs, found in a zygote are able to ge-n The p53 protein (encoded by the human geneTP53) is a piverate all embryonic and extra-embryonic cell types. Pluripotent otal tumor suppressor, designated ‘the guardian of the genome’ SCs, such as embryonic stem cells (ESCs) and induced pluripo- (5), because of its major role in maintenance of genomic st-a tent stem cells (iPSCs) have the capacity to differentiate into all bility and fidelity, thereby preventing cancer development6)(. cell types of the embryo proper. Multipotent SCs, such as me-s Indeed, recent studies demonstrated that elephants who are enchymal stem cells (MSCs) and hematopoietic stem cells pr-e resistant to cancer containing multiple copies of the p53 gene dominantly found in adult tissues are capable of contributing (7). Notably, p53 function is disrupted in the majority of human to various cell types usually derived from the same embryonic tumors. While mutations in the p53 gene were detected in more Abbreviations adipose-derived stem cell cancer stem cell embryonic stem cells gain of function hematopoietic stem cell induced pluripotent stem cell Li–Fraumeni Syndrome loss of heterozygosity mesenchymal stem cell stem cells Thus, while the possibility of prolongedin vitro propagation sets ESCs as valuable resources for regenerative medicine, it also may acquire ESCs with malignant potential, thereby comprom-is ing their safety for cellular therapies. Additional disadvantages of ESCs hindering their use for basic research and transplan-ta tion therapy are the possibility of tissue rejection and ethical concerns referring toex vivo destruction of human embryos. Characterization of iPSCs A decade ago Takahashi and Yamanaka introduced the groun-d breaking technology allowing the generation of iPSCs from mouse somatic cells by ectopic co-expression of the transcri-p tion factors Oct4, Sox2, Klf4 and Myc (OSKM)2(1,22). The availthan half of human cancer cases, its activity may be abrogated ability of man-made iPSCs provided new opportunities for the by alternative mechanisms in the other cases8(,9). development of autologous personalized medicine and disease In this review, we will describe the fundamental roles of wild modeling by overriding the ethical concerns associated with the type (WT) p53 and its mutants in the biology of the various SCs use of ESCs. Despite the initial observation that iPSCs succe-ss types. fully mimic ESCs, sharing a similar morphology, proliferation and pluripotent features, subsequent reports demonstrated that Bridging between SCs, CSCs and cancer the mRNA and miRNA expression signatures, epigenetic landscape as well as protein expression and protein phosphoryl-a Characterization of ESCs tion of iPSCs are distinguishable from ESCs2(3–26). Interestingly, ESCs derived from the inner cell mass of the blastocyst have iPSCs can be generated by introduction of variable combination the potential to give rise to cells that comprise the three germ of reprogramming factors, besides OSKM 2(7,28). For example, layers: endoderm, mesoderm and ectoderm 1(0). ESC can be it was recently found that ectopic expression of Sall4, Nanog, cultivatedin vitro for a prolonged period of time without lo-s Esrrb and Lin28 in mouse embryonic fibroblasts generated highing their pluripotency and self-renewal capacity, when suppl-e quality iPSCs more efficiently than other combinations of factors mented with reagents that prevent induction of differentiation including OSKM (29). In addition, it was shown that iPSCs could (11). The balance between ESCs self-renewal and differentiation be derived from a wide repertoire of cell types such as blood, is maintained by reciprocal regulatory networks governed by the stomach and liver cells, keratinocytes, melanocytes, pancreatβic transcription factors OCT4, SOX2 and NANOG that function as cells and neural progenitors 2(8). Of note is the observation that master regulators of ESCs identity12(,13). Preservation of proper cell origin influences the molecular and functional properties of genomic integrity is fundamental for maintenance of rapid pr-o the established iPSCs. This is attributed to the findings that iPSCs liferation and normal embryogenesisin vivo. As ESCs are the retain a transient epigenetic memory of their original somatic precursors of all adult organ systems, they must engage mech-a cells (30,31). Furthermore, high resolution studies indicated that nisms that prevent the propagation of mutations, generated during reprogramming the genetic integrity of the iPSCs may as a consequence of DNA damage, to their descendant mature be occasionally impaired. Indeed, iPSCs may gain genetic abersomatic cells. Indeed, it was found that compared with somatic rations including copy-number variations and protein-coding cells ESCs display lower mutation frequencies, as well as they point mutations 3( 2,33 ). Notably, it is now well-accepted the possess extreme sensitivity to DNA damage and exhibit el-e molecular mechanisms that underlie the generation of iPSCs are vated DNA repair capacity. Alternatively, ESCs with faulty DNA remarkably similar to those that are deregulated in cance3r4)(. may be eradicated from the self-renewing SC pool by induction Among these are dedifferentiation, gain of accelerated prolifer-a of either cell death or differentiation1(4). Nevertheless, multiple tion accompanied by senescence bypass, cancer-like changes in line of evidence indicated that long-term culturing of ESCs may metabolic profile and dynamic changes in epigenetic modificalead to gain of genomic abnormalities manifested by extensive tions that may acquire genetic alterations in cancer associated acquirement of point mutations, numerical and structural ch-ro genes. Accordingly, while on one hand the resemblance of iPSCs mosomal aberrations as well as variation of telomere length and generation and tumor development places the reprogramming alterations in epigenetic status, thereby conferring ESCs with technology as a valuable tool for cancer modeling, on the other tumorigenic potential1(5–17). hand the risk that iPSC may attain malignant characteristics Indeed, it has been already declared that ESCs share cellular hampers their safety for regenerative therap3y5(). and molecular phenotypes with cancer cells. Among these are rapid proliferation rate, lack of contact inhibition, high ac-tiv Characterization of adult SCs ity of telomerase, as well as high expression of oncogenes such Adult SCs usually reside in specific niches of self-renewing tisas c-MYC and KLF4 (17). Upon injection into immune-compro- sues including intestine, skin, muscle, fat, bone marrow and mised mice, bona fide ESCs give rise to teratomas, fully differe-n nervous system. Normally, adult SCs are quiescent, however, tiated benign tumors1(0). However, several studies have shown upon specific activation signals, they significantly promote that transplantation of culture-adapted ESCs resulted in g-en either normal tissue turnover or repair and regeneration foll-ow eration of aggressive tumors, such as teratocarcinom1a7(–19). ing injury ( 36 ). Notably, adult SCs number and function declines Concomitantly, it is was demonstrated that various types of with age in various tissues as a result of reduction in the act-iv poorly differentiated aggressive human tumors, exhibiting the ity of gate keeping tumor suppressors, DNA damage accum-u worst prognoses, display an enriched ESCs-like gene expression lation, changes in cellular physiology as well as environmental signature. Moreover, activation targets of Nanog, Oct4, Sox2 and changes in tissues ( 37,38 ). These, age-related changes in the c-Myc are observed more frequently in poorly differentiated function of SCs and other progenitors may contribute to agetumors than in well-differentiated tumor2s0(). associated degenerative diseases 3(7). Multipotent SCs such as MSCs and hematopoietic stem cells SC niche, a microenvironment that is crucial for the main-te also present in neonatal tissues such as umbilical cord and pl-a nance of SCs properties 4(4). centa. Regardless of their origin MSCs were shown to be able to Thirdly, it has been proposed that endogenous reprogramdifferentiatein vitro into chondrocytes, osteocytes, muscle cells, ming or residues of the embryo might serve as the source of adipocytes or even neurons and glia 3(9). Considering the afore- CSCs ( 46 ). mentioned concerns regarding the usage of ESCs and iPSCs for The major difference between normal SCs and CSCs lies clinical purposes, being multi-potent, hypoimmunogenic and on the ability to regulate stemness pathways including Wnβt-/ easy to isolate MSCs represent promising candidates for auto-lo catenin, JAK-STAT, TGF-β, Notch, Hippo, etc. In normal SCs, gous transplantation3(6). Nevertheless, one should bear in mind these pathways are tightly controlled with intact genetics and that duringin vitro culturing adult SCs including MSCs tend to epigenetics. In CSCs deregulation of these pathways along with undergo replicative senescence that is typically accompanied improper interactions between them may represent key events with progressive loss of proliferation and differentiation ability, for the propagation and pathogenesis of CSCs4( 9,53 ). morphological changes, high expression of tumor suppressors Investigation of CSCs in multiple tumor types have identified and increased telomere shortening 4(0). Moreover, despite the dozens of markers specific to these cells. However, several mar-k fact that adult SCs typically divide less frequently than plu-ri ers are shared between CSCs and normal SCs4(9). CD133 is one potent SCs, they are still prone to acquire chromosomal aber-ra of the most common cell surface markers used to identify CSCs tions during expansion in culture4(1). in breast, prostate, pancreas and lung tumor4s9( ,54 ). Additional surface molecules that were proposed for CSCs isolation are Characterization of CSCs CD34+/CD38−, CD44+/CD24−, CD44 alone or in combination with Carcinogenesis can be explained by two major models, the ‘st-o other antigens, and ephrin receptors (review in55[]). Aldehyde chastic’ model and the ‘CSCs’ model. According to the stochastic dehydrogenase 1 (ALDH1) is considered an internal marker for model all tumor cells result from clonal evolution through the normal and malignant SCs and progenitor cells. High ALDH1 acquisition of genetic mutations and epigenetic changes and activity is associated with chemo-resistance, aggressiveness are equipped with a similar tumorigenic potential. Whereas, the and poor clinical prognosis in many cancer types 4( 9,56 ). The CSCs model suggests that similarly to normal tissues, tumors main stemness transcription factors, Nanog, OCT4 and SOX2 show population hierarchy, where a subpopulation of CSCs has were also found to be highly expressed in CSCs ( 57 ). Recent evithe greatest tumorigenic activity compared to the other cells dence suggests that YAP1 and BMI1 are important intra-cellular comprising the tumor bulk 4( 2–44 ). CSCs are defined as rare, CSCs markers that endow SCs characteristics in multiple gast-ro malignant cells within the tumor, which are able to self-renew intestinal origin and head and neck cancers, respectively5(8,59). and differentiate and thus can restore the entire original cell Elucidation of the mechanism underlying CSCs features is pool of the tumor when injected into immune-deficient mice expected to permit development of novel cancer therapeutics ( 45 ). Being relatively quiescent, CSCs are resistant to chem-o targeting specifically CSCs, thereby eradicating the tumor and therapies, which mainly target rapidly dividing cells43(). The avoiding recurrence and drug resistance. hallmark traits of CSCs include their heterogeneity, inter-ac tCiSoCns walistohpmosiscersosentvhieropnrmopeenrttyaonfd‘rpolbausstticnietsys4’6,()w.Ihnichadidnictliuodne,s p53 in health and disease a number of features such as the capacity to perform efflux of Under normal physiological conditions p53 is maintained in low cytotoxic agents, resistance to oxidative stress and a fast DNA levels, due to continuous ubiquitination by its predominant ne-g damage repair, all of which facilitate drug resistance43( ,47,48 ). ative regulator, MDM2 that mediates its degradation by nuclear Furthermore, tumors with CSCs entity have been shown to p-os and cytoplasmic proteasomes6(0). Activation of p53 is driven by sess a higher propensity for metastasis in several cancers49(). various cellular insults including DNA damage, oncogene act-i The precise origin of CSCs is a debatable issue at present. vation, telomere erosion and hypoxia. These stress signals lead There are three main theories for the acquisition of the CSCs to p53 protein stabilization mainly by blocking its interaction phenotype ( 46 ). The first theory postulates that CSCs originate with MDM2 and by a series of post-translational modifications from malignant transformation of normal adult SCs that have ( 6,61 ). Once activated, aiming to eliminate the proliferation of undergone accumulation of epigenetic and genetic alterations. aberrant cells, p53 can determine the cellular fate by induction In this respect, a controversial report recently suggested that of cell cycle arrest, senescence or various types of cell death the variation in lifetime cancer risk for several tissues is p-re such as apoptosis, autophagy or ferroptosis, largely dependent dominantly attributed to random, ‘bad luck’ mutations arising on the severity and type of damage, as well as on the context during DNA replication in normal, noncancerous SCs maintai-n of cellular microenvironment 6( 2–65 ). The p53 protein structure ing that tissue’s homeostasis. Utilizing meta-analysis, Tomasetti consists of the amino-terminal transactivation domain, a p-ro and Vogelstein established that two-third of the human cancer line rich domain, a core DNA-binding domain, oligomerization types examined originate from SCs 5(0). However, the ‘bad luck’ and a carboxy-terminal regulatory domain66(). p53 functions hypothesis was later undermined by the study of Wuet al., who primarily as a sequence-specific tetrameric transcription factor, provided multiple lines of evidence, obtained from mathemat-i which regulates a multitude of downstream target gene6s7(,68). cal, epidemiological and molecular models, that cancer risk is In addition, accumulating data suggest that protein–protein profoundly influenced by extrinsic factors, while intrinsic risk interactions play an essential role in fine-tuning p53 activity at factors contribution to carcinogenesis is rather modes5t1,( 52 ). different levels of regulation 6(9,70). Notably, p53 may exert its The second theory pertaining the origin of CSCs relies on the effects not only inside a given cell, rather it can act in a nonnotion that tumors can acquire plasticity, which allows prog-eni cell-autonomous manner, thereby affecting the cell surroun-d tor or differentiated cells to undergo dedifferentiation acco-m ings (71–73). panied by epithelial–mesenchymal transition (EMT) and hence Although, the hallmark of p53 is its tumor suppressor act-ivi to become CSCs ( 44,46 ). Such phenotypic plasticity may result ties, ample data collected during more than three decades of from dynamic epigenetic changes regulated by signals from the research indicates that p53 functions a homeostatic regulator that coordinates a wide variety of additional cellular processes mice knocked in with either conformational mut-p53R172H or including metabolism (74,75), inflammation (76), aging (63,77), the DNA contact mut-p53R270H, equivalent to the codons 175 reproduction 7(8) differentiation and development 7(9,80), and 273 in humans. These mice displayed a wider tumor range regeneration 8(1) and others. and higher metastatic frequency compared with p53 null mice. The majority of p53 mutations found in human tumors result Correspondingly, mut-p53 homozygous primary cells derived from missense substitutions. As a consequence, a dysfunc- from these mouse models exhibited accelerated cell prolifer-a tional full-length p53 protein containing a different amino acid tion, DNA synthesis and high transformation potentia9l4(,95). in the mutation site is translated82(,83). Although analysis of Over the years of p53 research, great efforts have been de-di numerous human tumors indicated that mutations may occur cated to elucidate the mechanisms underlying mut-p53 GOF. in diverse locations along the p53 coding sequence, the distr-i Ample data obtained from variousin vitro and in vivo models bution of p53 mutations is not random. Most of the mutations indicated that the effect of mut-p53 GOF has been implicated in in p53 are concentrated within the sequence encoding for the various processes associated with tumorigenesis9(,92). These DNA-binding domain of the p53 protein, resulting in the mutant include deregulation of cell cycle and enhanced cellular prolifp53 (mut-p53) protein that is incapable of binding the respo-n eration ( 96,97 ), transformation9( 8,99 ), protection from cell death sive elements in the DNA of its classical target genes and exert and increased survival 1( 00,101 ), maintenance of chronic inflamtheir trans-activation9)(. Approximately 40% of p53 missense mation ( 102,103 ) invasion and metastasis 1(04,105) as well as mutations involve the following six ‘hot spot’ residues: R175H, drug resistance 1( 06,107 ). Of note, the various p53 mutants utilize G245S, R248Q/W, R249S, R273H and R282W (84). p53 mutations distinct mechanisms to execute their oncogenic GOF. Moreover, may be categorized into two main classes encompassing the the effect and the degree of oncogenic GOF exhibited by the p53 ‘DNA contact’ mutations and ‘conformational’ mutations. While mutants is dependent on the type of nucleotide substitution, ‘DNA contact’ mutations referred to substitutions in amino specific position in the p53 gene DNA sequence and the particuacids that involve sequence-specific DNA binding and is repr-e lar cellular conditions; thus further entangling the relationship sented by R248Q and R273H, ‘conformational’ mutations such as between mut-p53 genotype and phenotype8( 5,99,108 ). R175H, G245S, R249S and R282W cause modifications in protein Importantly, it has been generally considered that the ability structure, thereby compromising the capacity to bind DN8A5(). of mut-p53 to accumulate in the cells serves as a prerequisite Apparently, a considerable variation in the frequency of p53 for exhibition of its GOF 1( 09,110 ). In particular, the conclusion mutations is observed in diverse cancer types. For example, deduced from animal models was that mut-p53 proteins do not while it was found that only 10–20% of hematopoietic maligna-n accumulate in normal tissues, rather they are highly expressed cies exhibit p53 mutations 8(6), strikingly it was demonstrated predominantly in tumor cells 9( ,111,112 ). Similarly to the WT that p53 is mutated in 96% of high grade serous ovarian car-ci p53 protein, upon cellular stress mut-p53 undergoes post-tra-ns noma cases (87). lational modifications and becomes stabilized, however unlike Unlike p53 somatic mutations that promote the devel-op the WT protein, mut-p53 is not targeted for degradation by ment of sporadic tumors, p53 germline mutations are associated MDM2, rather remains constitutively active10(9). Interestingly, with the rare familial cancer predisposition termed Li-Fraumeni a recent elegant study using a novel mut-p53 mouse model Syndrome (LFS). LFS patients inherit one copy of p53 mutated expressing the R248Q hotspot mutation demonstrated that gene in every cell of their body and tend to develop early onset constant expression of stabilized mut-p53 is indispensable for of wide spectrum of cancer types, including bone and soft-tissue tumor maintenancein vivo. It was found that HSP90/HDAC6 sarcomas, acute leukemia, breast cancer as well as brain cancers chaperone machinery is a major determinant of mutp53 st-a such as glioblastoma and adrenocortical tumors88(,89). bilization. Ablation of mut-p53 or long-term HSP90 inhibition Similarly to the state of LFS patients, in the course of s-po that triggers mut-p53 destabilization led to diminished tumor radic tumorigenesis, a mutation in p53 gene initially arises only growth and extended survival1(13). This observation is consistin one of the alleles, resulting in p53 heterozygous cells that co-n ent with the oncogene addiction concept1(14) postulating that comitantly express both WT and mut-p53 proteins. In this case, although tumors display various genomic aberrations, some of mut-p53 may counteract the WT p53 tumor suppressor act-ivi them are dependent on single-specific oncogene, in this case ties via a dominant-negative mechanism, manifested by he-t mut-p53, which is crucial for both maintenance of the mal-ig ero-oligomerization with wild-type p53 through the C-terminal nant phenotype and cell survival. Additional relatively new tetramerization domain9(0). Eventually, during cancer progre-s mechanism suggested for mut-p53 both dominant-negative and sion, the remaining WT allele is lost, in a process termed loss of oncogenic GOF activities is its ability to aggregate into prion-like heterozygosity (LOH). Interestingly, recently performed genomic amyloid oligomers and fibrils. These aggregates sequester the and transcriptomic meta-analyses revealed that more than 93% p53 protein, thereby abolishing its transactivation function and of sporadic tumors harboring mut-p53 undergo p53 LOH91(). anti-proliferative effects1( 15,116 ). It was lately shown that p53 These findings support the notion that p53 is a recessive tumor protein aggregation promotes platinum resistance in ovarian suppressor and loss of the remaining WT allele is essential for cancer ( 117 ). Moreover, Soragniet  al. ( 118 ), have demonstrated tumor development. that a small cell penetrating peptide, ReACp53 designed to int-er Nowadays, it is well-accepted that besides abolishing the rupt mut-p53 aggregation into amyloid-like state rescues p53 tumor suppressive functions of the remaining WT protein, mut- tumor suppression in ovarian carcinomas. p53 proteins acquire new oncogenic functions that significantly All in all, this data suggest that depletion, destabilization or contribute to the malignant properties of the cells. This p-he disaggregation of mut-p53 might be potential therapeutic st-rat nomenon is referred as mut-p53 gain of function (GOF) 9(,92). egy to suppress tumor progression. The first report launching the concept of mut-p53 GOF was done bmyuDt-ipt5t3mperroett eainl.s(9in3t),owp5h3oddeefimcioennsttcrealltseednthhaantceidnttrhoedirutcutimono-roi f The role of p53 in ESCs genic potential. The most compelling evidence for mut-p53 GOF ESCs possess robust mechanisms to preserve their genomic was provided later following the establishment of transgenic integrity aiming to avoid transferring genetic alterations to their progeny. Therefore, it is not surprising that p53 ‘the guardian of Albeit the fundamental role of p53 in ESCs differentiation the genome’ was found to play an important role in maintaining programs, it was found that p53 null and p53 mutant knockedESCs genomic stability by regulating their differentiation and in mice are born viable and undergo quite normal development apoptotic pathways. with only minor abnormalities, although they develop a variety Unlike human ESCs in which p53 is expressed at very low of malignancies, later in postnatal life7(9,94,95,134–137). The levels due to negative regulators, HDM2 and TRIM24, that tr-ig relatively normal development of p53 knockout mice may be ger p53 degradation 1( 19,120 ), several lines of evidence indicated explained by existence of distinct compensatory mechanisms that mouse ESCs display high levels of the p53 protein, located executed by p53 family members, such as p63 and p73, as well mainly in the cytoplasm 1( 21,122 ). These increased p53 levels in as by other tumor suppressor genes7(9,80). Nevertheless, we undifferentiated mouse ESCs are mediated by low expression of found that p53-deficient mouse ESCs display a high incidence miRNA-125a and 125b ( 123 ). Interestingly, despite the abundance of karyotype abnormalities, including cells with increased nu mof p53 in ESCs, at first glance it seemed like p53 does not tran-s ber of chromosomes and translocations. Interestingly, mouse locate to the nucleus in response to DNA damage and does not ESCs knocked-in with conformational mut-p53 R172H remain induce apoptosis in ESCs ( 124 ). However, later in depth studies pluripotent and benign and have a relatively normal karyotype demonstrated that dependent on the type of cellular insult p53 compared with ESCs knocked out for p53. Strikingly, we disco-v may induce apoptosis of mouse ESC, both while it remains in ered that in these ESCs mut-p53 is stabilized into a WT p53 co-n the cytoplasm or translocates to the nucleus. For, instance, in formation. High-content RNA sequencing analysis of mut-p53 response to reactive oxygen species (ROS) cytoplasmic p53 tri-g ESCs indicated that in response to DNA damage p53 is transcr-ip gers mitochondrial-dependent apoptosis in mouse ESCs 1(25). tionally active and able to elevate the levels of classical WT p53 Alternatively, following ultra violet (UV) radiatioγni,rradiation or target genes, such as p21 and btg2. Utilizing mass spectrometrychemotherapy treatment p53 is able to enter to the nucleus and based technology we identified a network of proteins, including trans-activate its classical target genes including p21, mdm2, the chaperonin CCT complex, USP7, Aurora kinase, Nedd4 and puma and noxa and induce apopotosis 1( 21,122,126,127 ). In con- Trim24, that bind mut-p53 and thus may shift its conformation trast to differentiated cells, upon exposure to damaging agents to a WT form. Such conformational shift is a novel mechanism ESCs do not undergo the G1/S cell cycle arrest due to functional of maintenance of genomic integrity in ESCs 1( 38 ). In accorduncoupling of p53-p21 signaling (128). Apparently, ESCs possess ance with these findings it was reported by Trinidaedt al., that an alternative mechanism underlying p53-dependent mai-n proper folding of WT p53 is promoted by an interaction with the tenance of genetic stability, manifested by inducing the diffe-r CCT complex. Moreover, mut-p53 proteins unable to interact entiation into other cell types. More specifically, a land mark with CCT exhibited conformational instability, facilitated in-va report by Linet al., provided evidence that following DNA dam- sion and random motility that is similar to the activity of tumorage induced by UV radiation or doxorubicin treatment activated derived p53 mutants 1( 39 ). p53, phosphorylated on serine 315, suppresses the expression of Taken together, p53 significantly contributes to preservation the master regulator of pluripotentcy, Nanog, by direct binding to of ESCs integrity by inducing cell death or differentiation of cells its promoter, thereby inducing mouse ESCs differentiation12(6). with faulty genome. It might be suggested that the identified Interestingly, later by integrating global gene expression and proteomic network responsible for the stabilization of the mutcomputational analyses, Leeet al., discovered that phosphoryla- p53 protein into WT conformation, leading to the maintenance tion of p53 on serine 212 and serine 312 by Aurka leads to upreg-u of genomic integrity in pluripotent cells, can serve as the basis lated p53 activity that triggers ESC differentiatio1n29(). However, for development of future anticancer therapeutics. in human ESCs the mechanism underlying p53-dependent iinndduuccetiodniffeorfednitfifaetreionntiiantihounmisandiEfSfeCrse,npt5.3Itacwetayslastheodwonntlhysaitneto p53 as a barrier to iPSCs generation 373 activates expression of miR-34a and miR-145, which in turn Reprogramming of mature somatic cells into iPSCs is an inefficient repress the pluripotency factors OCT4, KLF4, LIN28A1(19). In process due to existence of multiple barriers. Thus, many stu-d concordance, a whole-genome study of p53-mediated DNA dam- ies were aimed to decipher the molecular mechanisms that may age signaling in ESCs indicated that in response to DNA damage, improve the quality and efficiency of reprogramming1( 40,141 ). As p53 affects the status of ESCs through activating differentiation- generation of iPSCs and malignant transformation share many associated genes and repressing ESCs-enriched genes (130). common characteristics3(5) it is not surprising that studies exa-m Interestingly, while it was observed that in human ESCs p53 l-ev ining the role of p53 found that WT p53 serves as a barrier of the els increase upon differentiation induction1(19), it was demon- reprogramming process. In accordance, p53 diminution markedly strated that in mouse ESCs differentiation led to a decrease in increased the efficiency of iPSCs generation 1( 42–147 ). For examthe levels of p53 and to a shift in its conformational status to the ple, Utikalet al. and Li et al., referred to the observation that onset mutant form, with a concomitant loss of functional activi1t2y1(). of senescence, which often results from increase in expression Of note, the pro-differentiation effect of p53 aiming to eliminate of the cell cycle inhibitors such as p16INK4a, ARF and p21Cip1 genomically damaged self-renewing cells is in line with the well as well as activation of p53, decrease reprogramming potential. It accepted notion that p53-dependent induction of different-ia was found that immortal fibroblasts deficient in components of tion in cancer cells blocks cancer progression 7(9,80). Moreover, the Arf-Trp53 pathway exhibited increased reprogramming kin-et it was observed that in somatic cells p53 may either promote or ics and a significantly higher efficiency than WT cells, endowing attenuate differentiation in a given cell type depending on c-el almost every somatic cell that normally fail to reprogram with the lular fate and distinct micro-environmental signals79(,131,132). potential to form iPSCs1(42,146). Notably, in contrast Hannaet al., In accordance, using a genome-wide study Lee et  al., revealed reported that the majority of the cells underwent reprogramming an anti-differentiation function of p53 in mouse ESCs through without depleting p53 or immortalizing the cells. They suggested directly regulating the Wnt signaling pathway. Thus suggesting that p53 deficiency only increased the kinetics of reprogramming, that p53 has a dual function also in mouse ESCs, regulating both without affecting the overall efficiency of the reprogramming pro-differentiation and anti-differentiation program1s33(). process (148). Others demonstrated that introduction of reprogramming DNA damage and chromosomal aberrations, that contribute to factors can activate the p53 pathway. Importantly, down--reg enhanced tumorigenic potential. Hence, while cells with abr-o ulation of p53 enabled fibroblasts to give rise to iPSCs using gated p53 pathway are not suitable for therapeutic applications, only Oct4 and Sox2, instead of OSKM group. However, constant recent findings demonstrate the plausibleness to utilize mutsuppression of p53 led to lower quality of iPSCs that displayed p53 expressing iPSCs in studying LFS associated malignancies. genomic instability 1( 43,144 ). Concomitantly, Marion et  al., reported that p53 is crucial to prevent generation of iPSCs -car The role of p53 in adult SCs rying various types of DNA damage, including short telomeres, DNA repair deficiencies or exogenously inflicted DNA damage. Ample reports have set p53 as a regulator of adult SCs diff-er Reprogramming in the presence of pre-existing, but tolerated, entiation. Apparently, p53 may either induce or suppress cel-lu DNA damage is aborted by the activation of a DNA damage lar differentiation not only by restricting proliferation, but also response and p53-dependent apoptosis 1( 45 ). by controlling master differentiation regulators. In this section, Later studies uncovering the mechanisms by which p53 we will briefly describe the most prominent studies highligh-t counteracts the reprogramming process showed that miR- ing the p53 regulation and function in neural stem cells (NSCs) 34a, a p53 target, plays an essential role in restraining somatic and MSCs. reprogramming by repression of pluripotency genes, including Nanog, Sox2 and N-myc (149). Among other p53-regulated ta-r The function of p53 in NSCs get genes, it was shown that p21, but not Puma played a partial Deranged neuronal development resulting in exencephaly in role in inhibition of iPSCs formation probably by attenuating cell approximately a quarter of p53 null embryos, was one of the division. Moreover, reactivation of p53 at any time point during initial evidences that p53 plays a prominent role in this process. the reprogramming process not only interrupted the formation This neural tube malformation is a result of either extensive cell of iPSCs, but also induced newly formed iPSCs to differentiate outgrowth or reduced apoptosis in the neural tissu1e3(7,156). (150). Interestingly, our findings indicated that p53 restricted NSCs are self-renewing cells in the nervous system that mesenchymal-to-epithelial transition (MET) during the early can generate both neurons and glia1( 57 ). p53 was shown to be phases of reprogramming and that this effect was primarily involved in the regulation of proliferation and differentiation of mediated by the ability of p53 to inhibit Klf4-dependent activ-a neural stem and progenitor cells, promotion of neuronal mat-u tion of epithelial genes (151). ration and axonal growth and regeneration following neuronal Furthermore, we found that mut-p53 R172H exhibited GOF injury, at large 1( 58 ). In particular, it was reported that p53 is by markedly enhancing the efficiency of the reprogramming expressed in the NSCs in the adult brain and negatively reg-u process, compared with p53 deficiency. Although mut-p53 iPSCs lates NSCs self-renewal (159). Furthermore, it was demonstrated maintained their pluripotent capacitiny vitro, following injection that in response to genotoxic insults such as X-ray or etoposide to immune suppressed mice, the reprogrammed cells expressing treatment, p53 maintained chromosomal stability in NSC1s6(0). mut-p53 lost this capability and gave rise to malignant tumors, Using transcriptomic profiling and functional studies of murine instead of teratomas generated by iPSCs expressing WT15(2). NSCs Zheng et  al., established that cooperative actions of p53 Later it was reported that distinct mut-p53 proteins have di-ffer and Pten down-regulating the expression of Myc are crucial for ent reprogramming efficiencies 1( 50 ). Interestingly, we observed the regulation of normal and malignant stem/progenitor cell that the reprograming kinetics of mut-p53 heterozygous (p53 differentiation, self-renewal and tumorigenic potential16(1). R172H/WT) mouse embryonic fibroblasts into iPSCs are comp-a Another mechanism suggested for the control of p53 in NSCs rable to WT p53 homozygous cells and similarly to WT p53 iPSCs, proliferation involves the inhibition of the activity and nuclear mut-p53 heterozygous iPSCs gave rise to teratomas. Unlike he-te localization of GLI1, which expression induces an increase in rozygous mouse embryonic fibroblasts that robustly underwent the number of NSCs. In turn, GLI1 represses p53 through activ-a LOH in culture, the majority of the iPSCs retained heterozygosity tion of Mdm2, establishing a homeostatic inhibitory loop that for prolonged periodin vitro. Nevertheless, a small percentage of normally controls the number of NSCs. Accordingly, primary iPSCs that underwent p53 LOH gave rise to malignant tumorins, glioblastoma SCs cultures expressing mut-p53 display increased vivo. This observation suggests that p53 LOH is attenuated d-ur levels of GLI1, consistent with GLI1-driven SCs expansion and ing the reprogramming process 1( 53 ). Intriguingly, in a recent tumourigenesis (162). Moreover, NSCs harboring mut-p53 di-s study Lee et  al., utilized iPSCs generated from fibroblasts of played impaired TGF-β induced growth inhibition, which conLFS patients heterozygous for mut-p53 G245D to investigate tributes to their increased proliferation rat1e63(). Alternatively, the role of mut-p53 in a model of osteosarcoma development it was reported that p53 regulates NSCs proliferation and -dif (154). The LFS-derived iPSCs were induced to differentiate first ferentiation via the BMP-Smad1 pathway and its target gene Id1. into MSCs and then into osteoblasts, which are the main cell Interestingly, Armesilla-Diazet al., demonstrated that differe-n type sustaining osteosarcoma1( 54,155 ). It was found that LFS- tiation of p53 knockout-derived neurospheres, that are enriched iPSCs-derived osteoblasts recapitulated osteosarcoma features for NSCs was biased toward neuronal precursors, compared to including defective osteoblastic differentiation and tumorigenic WT p53 neurospheres that were able to undergo differentiation potential. In addition, integration of global transcriptional and into astrocytes, oligodendrocytes and neurons16(0). However, computational analyses indicated that mut-p53 exerted GOF Zhou et al., reported that depletion of p53 alone could convert by suppressing H19 expression and its associated imprinted fibroblasts into astrocytes, oligodendrocytes and functional gene network component, decorin that is responsible for LFS- neurons. Apparently, knockdown of p53 upregulates neurogenic associated osteosarcoma development1( 54 ). transcription factors, which in turn boosts fibroblast-neuron All in all, p53 serves as a roadblock to reprogramming, faci-li conversion (164). Moreover, a recent study demonstrated that tating cell cycle arrest, senescence or cell death in cells with pre- suppression of p53, in conjunction with cell cycle arrest and existing DNA damage, thereby preventing the reprogramming appropriate cell culture environment markedly increases the of aberrant cells. Although p53 deficiency or mutation allow efficiency in the trans-differentiation of human fibroblasts to efficient generation of iPSCs, such iPSCs may carry persistent induced dopaminergic neurons by Ascl1, Nurr1, Lmx1a and miR124 (165). These findings underscore the complex regulation compared to their WT p53 expressing counterparts. Moreover, of p53 in the neuronal differentiation process. p53 may diffe-r MSCs lacking p53 displayed genomic instability, changes in entially regulate a the plasticity of diverse cell types, i.e. NSCs the expression of c-myc and anchorage-independent growth and fibroblasts, depending on the cell of origin and extracellular (166,177). Interestingly, it was shown that MSCs lacking the p21 surroundings. gene and bearing only one functional allele of WT p53 underwent p53 LOH during prolonged culturing that conferred them The role of p53 in MSCs with tumorigenic potential, manifested in formation of sar-co Multipotent MSCs reside mainly in the bone marrow but can mas upon injection into immune-compromised mice 1(78). In also be found in other tissues including bone, adipose tissues, agreement, our data showed that MSCs heterozygous for mutmuscle and many more. MSCs play an important role in no-r p53 predominately undergo p53 LOH conferring them with the mal tissue turnover as well as in regeneration and repair f-ol high carcinogenic potential 1( 53 ). lowing injury ( 36,39 ). Nonetheless, it was reported that MSCs Taken together, multiple mechanisms have evolved to ensure may contribute to tumor development. They were suggested proper p53 regulation of the various aspects of MSCs biology to be a major component of tumor stroma able of modifying including differentiation, proliferation as well as immune-mo-d tumor growth, or alternatively serve as an origin of certain ulatory functions, to avoid deterioration towards carcinogenesis. sarcomas (166). playEsviadecnecnetsroabltraoilneeidn ftrhoembnioulmogeyrooufsMsStCuds.ieFsoirnedxicaamteplteh,aMtSpC5s3 The role of p53 in CSCs derived from p53 knockout mice exhibited an enhanced di-f Accumulating data indicates that CSCs and p53 mutant proteins ferentiation towards the osteogenic and adipogenic lineages, endow the cells with common oncogenic features including suggesting that p53 plays a negative regulatory role in these accentuated tumorigenic potential, protection from cell death processes (132,167–169). However, it was shown that in other and drug resistance, capacity to invade and metastasize, as well circumstances p53 may facilitate osteogenic differentiation. For as cancer-associated metabolic profile 1(06,178). Additionally, instance, more than 20  years ago Radinskyet  al., have shown gain of CSCs phenotype via acquisition of plasticity and de-d-if that introduction of WT, but not mut-p53, into p53-deficient ferentiation process has been associated with EMT4(4,45), which osteosarcoma cells was associated within vivo induction of ter- has been also shown to underlie the oncogenic GOF of mut-p53 minal differentiation and apoptosis that inhibited progressive (179–181). Moreover, a compelling evidence linking mut-p53 GOF growth of metastases 1(70). Utilizing gel shift and chromatin and dedifferentiation is the association of mutations in p53 with immune-precipitation assays, a more recent study corroborated poorly differentiated tumors, such as thyroid carcinoma1s8(2), this observation, demonstrating that introduction of WT p53 gliomas (183), squamous cell carcinoma (184) and ovarian carc-i into osteosarcoma cell line resulted in a direct transactivation nomas (185). Bearing these observations in mind, it is tempting of the osteocalcin expression, which is produced in the terminal to speculate that mut-p53 GOF may facilitate the rise of cells stages of osteogenic differentiation 1(71). These observations exhibiting CSCs characteristics. In accordance, it appears that suggest that p53 may alter its effect on osteogenic differ-en several studies have contradicted the statement that mut-p53 tiation depending on a specific cellular state and tumorigenic stabilization is unique for cancer cells. Accumulation of mut-p53 potential. was observed in sub-populations of non-cancerous cells such as Numerous evidence derived from in vitro and in vivo models human keratinocytes 1(86), human epithelial cells of distal fa-l support the notion that p53 plays a restrictive a role in the di-ffer lopian tubes (187) as well as in a subpopulation of highly proli-f entiation towards the adipogenic lineage in MSCs as well as in erating cells in the small intestine of mut-p53 knocked-in mice other more committed adipogenic precursors79(,172). Adipose- (188). Thus, it might be plausible that upon further perturbations derived stem cells (ASCs) possess multilineage differentiation and appropriate environmental conditions these cells will serve capacity that offers the potential for the repair or regeneration as primary candidates to acquire CSCs phenotype. of various tissues 1(73). Interestingly, while the examination of In line with its tumor suppressor activities, WT p53 was p53 expression levels in proliferating ASCs and terminally d-if shown to compromise CSCs features by repression of CSCs ferentiated adipocytes did not reveal any differences1(74), we markers such as CD133 (189) and CD44 (190) either by direct have shown that knockdown of p53 in hASCs resulted in a si-g binding to the promoter or indirectly, through inducing its target nificant increase in their adipogenic differentiation capacity, gene miR-34a, respectively. Whereas, alterations in the p53 st-a supporting the restrictive effect of p53 on white adipogenic d-if tus have been found to contribute to CSCs properties in various ferentiation 1(31). cancer types. Several examples are described below. Within the tumor microenvironment, MSCs acquire the By using mouse models Fleshkin-Nikitin et al. (191), identicharacteristics of carcinoma-associated fibroblasts and support fied a cancer-prone SCs residing at the junction area of ovarian tumor growth. p53 was found to regulate MSCs function in a surface epithelium. These SCs expressing the SCs markers such tumor microenvironment through different mechanisms. One ALDH1, LGR5, LEF1 and CD133 gained enhanced transformation of the mechanisms entails p53 dependent inhibition of MSCs potential following alteration of p53 and Rb1 status and were motility towards tumor cells by decreasing the transcription of suggested to constitute the cells of origin of epithelial ovarian the chemokine CXCL12 (also known as SDF1), which is necessary carcinomas. Likewise, Wang et  al., revealed that initiation of for MSCs migration in response to tumor cells17(5). Another glioma formation is associated with the appearance of NCSs mechanism involved p53-mediated reduction of the levels of expressing detectable level of mut-p53 proteins in the subve-n iNOS, leading to diminished immunosuppressive capacity of tricular zone of the brain and the subsequent expansion of mutMSCs and as a consequence attenuated tumor growth17(6). p53-expressing Olig2+ transit-amplifying progenitor-like cells Lack of p53 in MSCs was also shown to augment their ce-l (192). Others reported that NSCs transformation may result from lular growth as manifested by increased proliferation rate, a inhibition of oxidative metabolism that leads to p53 genetic shorter doubling time and enhanced colonies formation capa-c inactivation. Moreover, p53 mutations correlate with alterations ity, loss of p16 expression and lack of senescence response, as in mitochondrial metabolism and genomic instability in primary glioma-initiating NSCs 1(93). Alternatively, it was demonstrated therapy. One of the major compounds related to mut-p53 re-ac that brain CSCs generation can result from p53-deficient mouse tivation is CP-31398 that can stabilize the DNA-binding core astrocytes either by expression of oncogenic Ras 1(94) or via domain of several mut-p53 proteins, induce conformational Nanog-induced dedifferentiation 1(95). change and restore p53 transcriptional activity and function Several studies have provided evidence from mouse models (202). Additional prominent compounds refolding mut-p53 into that tumors with sarcoma features can arise from iPSCs, MSCs WT p53 conformation are PRIMA-1 and its analog PRIMA-1MET or poorly differentiated osteoblasts bearing at least one end-og that is currently in clinical trials. The mechanisms underlying enous mut-p53 allele (152–154). In addition, it was observed that their action involve the conversion of these compounds to pro-d spontaneous transformation of MSCisn vitro is accompanied by ucts, which form adducts with thiol groups in the DNA-binding concomitant gain of p53 mutation in the DNA binding domain domain of p53 mutant proteins, leading to restoration of WT and CSCs-like characteristics1(96). In line with these observa- activity and induction of apoptosis in tumor cells20(1,203). tions, it was shown that in human osteosarcoma cells, mut-p53- In this respect, using innovative approach of screening phage R248W/P72R oncogenic GOF promoted cancer stem-like features display libraries combined with deep sequencing methodology reflected by high proliferation rate, sphere formation, clon-o we have recently obtained a large database of mut-p53 pote-n genic growth, high migration and invasiveness. Furthermore, it tial reactivating peptides. The identified lead peptides were strongly increased the levels of stemness proteins1( 97 ). able to stabilize the correct conformation of mut-p53, endo-w In the mammary gland, the physiological function of p53 is ing mut-p53 with WT p53-like activities and cause regression to maintain a constant number of SCs by imposing an asym- of mut-p53-bearing tumors in several xenograft models. Thus metric mode of self-renewing divisions. It was demonstrated, suggesting that these peptides might serve as novel agents for that loss of p53 in mammary SCs increase the frequency of sy m- human cancer therapy 2(04). metric cell divisions, thereby providing them with CSCs self- As treatment of tumors with multiple independent mod-ali renewal properties that contribute to mammary tumorigenesis ties appears to yield beneficial anti-tumor responses, intri-gu (198). In addition it was shown that the p53 isoformΔ133p53β, ingly Zhang et  al., explored the possibility to target CSCs with lacking the transactivation domain, promotes CSCs potential p53 modulators. In this study, CSCs exhibiting high ALDH1 act-iv in breast cancer cell lines. This is manifested by the expre-s ity from human breast, endometrial and pancreas carcinoma sion of the key pluripotency factors SOX2, OCT3/4 and NANOG cell lines expressing either WT or mut-p53 were treated with in a p53 Δ133p53β-dependent manner. Notably, treatment with CP-31398 and PRIMA-1. The small molecule compounds siga cytotoxic anti-cancer drug, further increased CSCs formation nificantly reduced CSCs content and sphere formation by these via p53 Δ133p53β, thus potentially increasing the risk of cancer cell lines in vitro. In addition, these agents were more effective recurrence (199). against CSCs compared to the chemotherapy such as cisplatin Collectively, perturbations in the p53 pathway seem to co-in and gemcitabine (205). cide with the generation of CSCs both by malignant transf-or Considering the wide repertoire of p53 mutations in the mation of normal SCs as well as by acquisition of plasticity by various cancer types and the distinct markers of CSCs, future mature cells. Hence, providing a strong rationale for combining development of therapeutic strategies should be directed therapeutic strategies targeting CSCs with compounds which towards the concepts of precision medicine and involve comcan deplete mut-p53 and restore wild-type p53 activity. bination therapy encompassing drugs targeting CSCs and mutp53. Design of effective and safe compounds that exclusively Therapeutic strategies target mut-p53 as well as therapeutic strategies that specifically hinder CSCs will permit improving clinical efficacy of cancer therapy. Being a cornerstone of the carcinogenic process, CSCs were found to play fundamental roles in distinct stages of ca-n cer development and metastasis, acquiring tumors with drug resistance and sustaining cancer recurrence. To permit more Concluding remarks efficient cancer treatment, novel therapeutic approaches were Maintenance of genomic fidelity is of paramount importance for designed aimed to eradicate CSCs. Most common therapies SCs integrity to allow normal development, proper different-ia aimed to hamper CSCs regeneration and cancer relapse include tion and regeneration, to assure cancer prevention. Therefore, targeting the specific surface markers and signaling pathways SCs exert stringent mechanisms that prevent the propagation underpinning the CSCs properties, as well as induction of of genetic alterations to the descendant cells. Ample accum-u CSCs apoptosis and differentiation. However, since CSCs and lated data indicate that p53, the major tumor suppressor gene, normal SCs share some similar characteristics, albeit holding plays a fundamental regulatory role in SCs fate decisions with great promises for cancer therapeutics, some of these therapies an aim to eradicate aberrant SCs form the self-renewing SC against CSCs may be not specific, thereby destroying normal pool. Importantly, mutations in the p53 endow it with both loss SCs niches (200). of tumor suppressive function as well as with oncogenic GOF p53, the most frequently mutated gene in human cancers, that greatly facilitates tumorigenesis. In ESCs, following DNA has been an attractive target for cancer therapy for many years. damage, WT p53 induces either cell death or differentiation of Numerous strategies targeting p53 have been developed inclu-d cells with faulty genome. As carcinogenesis and reprogramming ing gene therapy to restore WT p53 function, drugs aimed at share common molecular mechanisms, p53 serves as a ba-r disruption of p53-MDM2 interaction, as well as small-molecule rier along the reprogramming process, preventing generation compounds that specifically target mut-p53, thereby elimin-at of iPSCs from suboptimal somatic cells. Furthermore, p53 may ing its oncogenic GOF by depleting it or restoring WT p53 tumor either induce or repress differentiation of the various adult SCs suppressive activities 2(01). Most of the above approaches were and precursor cells, depending on the cell of origin and microdeveloped either by rational design or by screening of small environmental signaling. 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Molchadsky, Alina, Rotter, Varda. p53 and its mutants on the slippery road from stemness to carcinogenesis, Carcinogenesis, 2017, 347-358, DOI: 10.1093/carcin/bgw092