This CH2 domain contains a very crucial S36 residue which is phosphorylated in response to oxidative stress and plays a role in apoptosis. Whereas p52Shc and p46Shc are ubiquitously expressed, p66Shc shows constrained expression. This adaptor protein has been shown to be involved in mediating and executing the post effects of oxidative stress and increasing body of evidence is pinpointing to its role in carcinogenesis as well. It shows proto-oncogenic as well as pro-apoptotic properties. This multitasking protein is involved in regulating different networks of cell signaling. On one hand it shows an increased expression profile in different cancers, has a positive role in cell proliferation and migration, whereas on the other hand it promotes apoptosis under oxidative stress conditions by acting as a sensor of ROS (Reactive Oxygen Species). This paradoxical role of p66Shc could be attributed to its involvement in ROS production, as ROS is known to both induce cell proliferation as well as apoptosis. p66Shc by regulating intracellular ROS levels plays a crucial role in regulating longevity and cell senescence. These multi-faceted properties of p66Shc make it a perfect candidate protein for further studies in various cancers and aging related diseases. p66Shc can be targeted in terms of it being used as a possible therapeutic target in various diseases. This review focuses on p66Shc and highlights its role in promoting apoptosis via different cell signaling networks, its role in cell proliferation, along with its presence and role in different forms of cancers. The Shc family of proteins consists four members ShcA, ShcB, ShcC and ShcD, of which the best characterized to date is ShcA ,2. ShcA,p46Shc and p52Shc are ubiquitously expressed, while p66Shc is expressed at different level in various tissues. The CH1 domain of p46/p52Shc has three tyrosine residues which are phosphorylated in response to a number of cell surface ligand activated receptors. p52Shc binds to phosphorylated receptors via the PTB domain or the SH2 domains in response to growth factors, and is phosphorylated on these tyrosine residues  within the CH1 regions. Grb2 (Growth Factor Receptor-bound Protein 2) forms a complex with SOS (Son of Sevenless) protein. The Grb2/Sos complex is recruited by the phosphorylated p52/p46Shc proteins. This Grb2/Sos complex is then docked onto these phosphotyrosine residues, via direct interaction with the Grb2-SH2 domain, which ultimately leads to the activation of Ras proteins . This coAnother adaptor protein E3b1 together with Eps8 also plays a role in regulating the phosphorylation, reducing ubiquitilation and increasing the stability of p66Shc protein through Rac1 . Sos1 cain vivo has been observed to increase metabolic rate as well as decrease fat mass and resistance to diet-induced obesity. p66Shc generated ROS regulates the effect of insulin and may lead to acceleration of aging by favoring fat deposition and as a result fat related disorders [p66Shc is also a part of a signal transduction pathway that is activated by increased intracellular ROS, leading to apoptosis. The oxidative stress resistance and lifespan of a cell and an organism is increased by mutation of this pathway. The accumulation of oxidatively damaged macromolecules occurs in virtually all aging mammals . In C. eisorders .Furthermore, depletion of p66Shchas been shown to lead to Warburg effect, causing enhanced glycolysis and increased allocation of glucose-derived carbon into anabolic metabolism. This role of p66Shc has been observed in mice that are deficient in p66Shc. These p66Shc deficient mice show resistance to diabetes and obesity. This altered metabolism was seen to be mediated by mTOR . This indicates towards an inhibitory role of p66Shc in anabolic metabolism, unlike other isoforms of Shc . Stimulap66Shc has also been shown to contribute to EAE  induced neuronal damage. It does so, most likely, through the opening of PT pore that triggers mitochondrial swelling and leads to neurodegenerative stress . In β-amp66Shc has a unique CH2 region at the NH2 terminal. This CH2 region is of around 110 amino acids and, like the CH1 domain, is rich in glycine and proline residues. The CH2 region contains the unique and all important serine phosphorylation S36 and S54) sites . The CH24 sites .Under oxidative stress the S36 residue of p66Shc is phosphorylated. Treatment with an iron-containing porphyrin, hemin increased the phosphorylation of p66Shc at the S36 residue. In hemin treated K562 erythroleukemic cells p66Shc was transcriptionally activated through the ARE (Antioxidant response element)-Nrf2 (NF-E2-Related Factor 2) pathway . In humaTill now most of the studies have demonstrated that p66Shc shows a pro-apoptotic and pro-oxidative role. Up to 30% extension in lifespan was seen in p66Shc knockdown mice. These knockdown mice also showed better handling of ROS and oxidative stress ,36-38. TThe S54 site in the CH2 domain of p66Shc is important for its stability . The pro2S inhibited the mitochondrial ROS production and as a result, phosphorylation of p66Shc and lead to sulfhydration of p66Shc on C59 residue [2O2 was also seen to be mediated by p66Shc [Recently it was seen that in SH-SY5Y cells or in the mice cortex, exogenous H residue . ROS arey p66Shc . Many sty p66Shc . The assy p66Shc .2O2. All these studies point to the fact that p66Shc is needed for bringing about apoptosis. Phosphorylation of p66Shc on the S36 residue has been shown to have a pro-apoptotic effect in different cell lines. Upon expression of p66S36A mutant into these p66Shc-/- cells, they are still not able to restore the pro-apoptotic responses [The increased levels of ROS act as the trigger to interfere with many cellular processes. These triggers result in inhibition of cell proliferation and induction of apoptosis. Apoptosis is the process of programmed cell death that takes place in multicellular organisms and comprises of many cellular events like cellular blebbing, nuclear fragmentation, chromosomal DNA fragmentation and ultimately cell death. p66Shc is known to have a role in apoptosis triggered by oxidative stress. The affirmation to this has been seen in a number of studies. Oxidative stress gives a stimulus to mouse thymocytes, peripheral blood lymphocytes and splenic T-cells and these cells then attain the ability to express p66Shc -48,50,51esponses -30.The gene encoding p66Shc has also attracted major interest in aging research in the recent years since it plays a vital role in apoptosis induced by oxidative stress. p66Shc is known to have a crucial role in the regulation of intracellular ROS levels . The accROS can either originate from exogenous sources such as ultraviolet and ionizing radiations, or it can be endogenous . ROS has been implicated in the damage of cellular proteins, lipids and DNA. This oxidative cellular damage is apparently the main origin of aging. It causes mutations and deletions in both nuclear as well as mitochondrial DNA ,44,53. VThe p66Shc-/- fibroblasts cells have altered bio-energetic properties vis-à-vis decreased resting and maximal oxidative capacity. This suggests that deletion of p66Shc may extend lifespan by shifting metabolic energy away from oxidative and toward glycolytic pathway . One can2O2 and UV. p53-/- MEFs also show resistance to UV-induced and H2O2-induced apoptosis [The tumor suppressor p53 is critically involved in oxidative stress-dependent apoptosis. p53 has a role in mediating the oxidative stress response of p66Shc, since p53 is up-regulated upon treatment with Hpoptosis . Increaspoptosis . p66Shc poptosis   help the cells to sense their location as well as that of the neighboring cells. When these cell-ECM interactions are incorrect, apoptosis takes place. This kind of location specific apoptosis is termed anoikis. Anoikis is a special case of programmed cell death that is initiated by cell detachment instead of the normal pro-apoptotic signals . AnoikisCells that lack p66Shc have been shown to escape anoikis. Anoikis in these cells was restored with the reexpression of p66Shc. p66Shc restores anoikis via RhoA activation. Transient as well as stable transfections with p66Shc lead to cell death in detached cells, but not in adherent cells. In contrast to apoptosis caused by oxidative stress, this ability of p66Shc to confer anoikis properties is not dependent on S36 phosphorylation of p66Shc . In addiCell proliferation, migration and adhesion are the main properties of a tumor cell. Tumor cells proliferate, migrate and adhere to target tissue. These steps allow the tumor cell to obtain metastatic phenotype. Cell proliferation and migration depend on the signals transmitted by growth factors and adhesion proteins . These pp66Shc also plays a crucial role in proliferation of epithelial cells. The processes of cell proliferation and apoptosis have to be well balanced. The balance of these two processes is governed by signal transduction machinery . Any imbARF1 has been found in complex with Grb2 and p66Shc upon EGF stimulation of the basal like breast cancer MDA-MB-231 cell line. The small GTPases ARF1 and ARF6 have been shown to be activated downstream of the EGFR and act as key regulator of growth, migration and invasion of breast cancer cells . Estrogep66Shc protein levels were seen to be closely correlated to the growth rate of prostate cancer cells. p66Shc protein levels were also seen to be significantly higher in prostate cancer adenocarcinomas as compared to the adjacent benign glandular cells ,97. p66SA consistent increase in the p66Shc protein levels were observed in ESCC  and EAC . The controls of ESCC showed a lower basal level of p66Shc expression . In the 2O2 [The expression of p66Shc was seen to be increased in all kinds of proliferating thyroid tissues, but not in the normal thyroid tissue of the same patient . This da2O2 .Aiolos, which is often expressed in lung cancers and correlated with reduced patient survival, has been seen to be inversely correlated with the expression of p66Shc in lung cancer tissue as well as in single cells . On the In ovarian cancer cells the level of ErbB-2 expression positively correlates with the level of p66Shc. ErbB-2 is a prognostic marker for ovarian carcinoma . In ovarIn colon cancer tissues, cancerous cells show higher levels of p66Shc proteins as compared to the adjacent non-cancerous cells . p66Shc A recent study has shown the regulatory role of p66Shc-derived mitochondrial ROS. The p66Shc-derived ROS has a regulatory role on growth factor signaling by induction of protein tyrosine phosphatase (PTP) oxidation. It was seen that p66Shcdeletion and down regulation lead to decrease in PDGF-induced signaling and migration, by decreasing PDGF-induced PTP oxidation . In summp66Shc being a candidate protein that controls both cell death as well as cell proliferation and plays a crucial role in regulating longevity and cell senescence, by regulating intracellular ROS levels. These counteracting properties of p66Shc make it a perfect candidate for further studies in various cancers and aging related diseases. p66Shc can be targeted in terms of it being used as a possible therapeutic target in various diseases, by figuring out how to use p66Shc protein for balancing the ROS levels in the cells and tipping the scales in our favor.


N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.p53 protein has about thirty phosphorylation sites located at the  In adp53 phosphorylation ,7 at almGiven the functions controlled by p53 phosphorylation, it is expected that the specificity and dynamics of p53 phosphorylation are carefully regulated, and mutations would affect p53 phosphorylation pattern and function. Phosphorylated p53 mutants were found to accumulate in tumor tissues ,28. p53 C-terminal homo-oligomerization domain and becomes phosphorylated predominantly at Y99, and also at Y121 and Y240 . It. It46PDDF1B1/p62 .N-terminal transactivation domain (TA), a DNA-binding core domain, a C-terminal tetramerization and a regulatory domain. In addition, p63 and p73 also have a sterile alpha motif domain (SAM) at the end of the C-termini. Most of the N-terminal TA domain and C-terminal regulatory domain are highly flexible and have no well-defined structure. Therefore, p53 can also be classified as a typical intrinsically unstructured protein [The structures of p53/63/p73 contain an  protein ,100, whi protein ,101. The protein ,103.It was suggested that natively disordered proteins are more likely to be associated with cancer . p53 is p53 gene transfer in human glioma correlates with site-specific phosphorylation [Gene therapy to restore p53 function is among the clinical approaches currently under investigation . For exarylation ,104, sugrylation . It is krylation . While siYj=i+1, XiYj=i+2, and XiYj=i+3, which are equivalent to the dipeptide XY, tripeptide X × Y, and tetrepetide X ×× Y motifs, respectively. Then we calculate the propensity of amino acid pair association by normalizing the frequencies of the overall counts of individual amino acid in the p53, p63, or p73 families:XiYj is the propensity of amino acid pair association in XiYj=i+1, XiYj=i+2, and XiYj=i+3 positions, respectively. NXiYj is the total number amino acid pair counts in XiYj=i+1, XiYj=i+2, and XiYj=i+3 positions in all sequences in each family, respectively. The NX and NY are total numbers of amino acid type (X and Y) in all sequences in each p53, p63, or p73 families [XiYj is similar to that used by Vonderviszt and Simon [XiYj/(NXY × PX × PY) to measure the dipeptide propensity . PXiYj in equation 1 changes more smoothly with the variation of NX and NY in the calculation of the dipeptide propensities across evolution [For each sequence in a protein family, we count the two amino acids (X and Y) in the positions of Xfamilies . Thus, and Simon , who usevolution .XiYj by adding PXiYj and PYiXj for non-diagonal elements in the correlation matrix.We calculate the degenerate dipeptide pair correlation propensity DPThus, we do not distinguish between XY and YX and label such amino acid combinations X/Y, and the propensity X/Y is obtained from the combined XY and YX counts.Disorder propensities were calculated using the webserver FoldUnfold . The aveWe use ClustalX 2.0  to alignp53 gene therapy.The existence of almost thirty phosphorylation sites in p53 poses a considerable evolutionary pressure on p53 to selectively bind to specific kinase with the right phosphorylation motif to respond to correct phosphorylation signals. What are the sequence and structural features in p53 phosphorylation motifs? Why and how p53 phosphorylation sites are rarely mutated in human cancer? In our study, we focused on the phosphorylation sites and nearby flanking residues and computationally examined the consensus phosphorylation motif pattern, amino acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer-related mutations observed within the phosphorylation motifs. Among the many factors potentially contributing to the safeguarding mechanism against mutations in p53, we found two patterns that appear unique and correlated with p53 mutations. p53 proteins have high propensities for acidic amino acids adjacent to potential phosphorylation sites. The negative charge near a phosphorylation site might make a mutation of the phosphorylation site less deleterious, since the motif already has a negative charge and may still have certain electrostatic interactions with other binding partners. The above conclusion is consistent with experimental observations. p53 phosphorylation motifs are mostly disordered. Even though cancer-related mutations largely decrease the disorder propensity of the phosphorylation motifs, the disordered nature of phosphorylation motifs might still help to compensate for mutational effects on p53 phosphorylation. Our computational results suggest that engineered acidic amino acids adjacent to potential phosphorylation sites could be a potential strategy in


Drosophila have unraveled intriguing roles of Dmp53 in promoting cell division in apoptosis-induced proliferation, enhancing fitness and proliferation of the winner cell in cell competition and coordinating growth at the organ and organismal level in the presence of stress. In this review, we describe these new functions of Dmp53 and discuss their relevance in the context of carcinogenesis.The canonical role of p53 in preserving genome integrity and limiting carcinogenesis has been well established. In the presence of acute DNA-damage, oncogene deregulation and other forms of cellular stress, p53 orchestrates a myriad of pleiotropic processes to repair cellular damages and maintain homeostasis. Beside these well-studied functions of p53, recent studies in  The p53 protein is the product of the tumor suppressor gene, TP53. It is a member of the p53 superfamily proteins that comprise TP53, TP63 and TP73. p53 is mutated or inactivated in more than half of human cancers. Early works on p53 have elucidated its canonical function in response to DNA damage. Specifically, in the presence of mild stress or damage signal, p53 blocks cell cycle progression and activates DNA repair machinery to promote cell survival and maintain genome integrity. However, when the damage is extensive, p53 drives cellular senescence or irreversible apoptosis to eliminate the potentially oncogenic cells with genomic instability and therefore reduces the risk of tumorigenesis , 2. In aDrosophila is instrumental to study the pleiotropic functions of p53 in both stress response and development [Drosophila melanogaster p53 (Dmp53) and found it to be the only member of the p53 family proteins in flies [Drosophila studies showed that after irradiation treatment, Dmp53 mediates apoptosis but not cell cycle arrest [C. elegans, the p53 homologue, cep-1 still harbors a SAM domain. However, studies from Banerjee and col. have challenged this view as Drosophila p53 is able regulate cell cycle upon energy deprivation: the “tenured” mutation disrupts the mitochondrial electron transport chain and causes reduced cellular ATP level, which leads to a G1/S checkpoint arrest that requires Dmp53 [Drosophila p53 carries the ancestral functions of all p53 family members . This ies Dmp53 . Specifies Dmp53 . These rDrosophila p53-mediated apoptosis, DNA repair and homologous recombination, but also were among the first to assign physiological and developmental functions to p53 [Since the cloning of Dmp53, studies in the fly primordial germ cells, imaginal discs and adult photoreceptors have not only elucidated the detailed mechanisms underlying s to p53 –19. Reces to p53 .Drosophila have unraveled intriguing roles of Dmp53 in promoting cell division in apoptosis-induced proliferation, enhancing fitness and proliferation of the winner cell in cell competition and coordinating growth at the organ and organismal level in the context of stress , which in turn induce immediate and local proliferation in the healthy surrounding cells [Drosophila [In response to damage or injuries in the developing tissue, apoptotic cells promote cell proliferation in the surrounding tissue to repair and regenerate , 23. Apong cells –26. Thisosophila –29 and iosophila . AiP is osophila .rpr or hid, but apoptosis is concomitantly inhibited by the expression of the caspase inhibitor p35 [AiP was first studied using “undead cells”, which are generated by subjecting the cells to a pro-apoptotic signal, such as itor p35 , 29. In itor p35 , 32. Theitor p35 –35. Impoitor p35 , 36. In itor p35 . This paCnidaria hydra [There is evidence that such Dmp53-controlled proliferation and AiP program are conserved throughout evolution. In the planarians, p53 regulates proliferation and self-renewal in adult stem cell lineages . Similaria hydra , in thatia hydra . Interesia hydra . In thisDrosophila p53 regulates multiple targets essential for apoptosis, DNA repair and proliferation [Drosophila, there are four annotated transcripts for Dmp53, which leads to the expression of two detectable protein isoforms: Dp53 and DΔNp53  that contains a putative p53 binding site, the authors showed that varying Dmp53 protein levels regulate N gene transcription. This study corroborates previous mouse studies showing that p53 binds Notch1 promoter and that p53 silencing resulted in reduced Notch expression [In this issue of Apoptosis, Busturia and col. have examined the contribution of Notch (N) signaling to AiP in relation to DΔNp53 . The autpression , 46. Thepression .dronc- and dmp53-dependent manner; while in differentiating tissues such as the posterior part of the eye disc, Hh is preferentially activated in the dying cells via the non-apoptotic activation of the effector caspases Drice and Dcp1 [While overexpressing DΔNp53 activates N and Wg expression in the developing wing, it is unclear how N, Wg, Dpp and Hh contribute to the different AiP paradigms. It was proposed that distinct programs of AiP are activated depending on the cellular state , 26. Forand Dcp1 . Thus, ddronc mutants [Drosophila p53, the induction of proliferation by Dmp53 is prompted by apoptosis upon stress. In the classic DNA damage response, Dmp53 rapidly activates rpr expression and hence the apoptosis cascade. A recent study by Shlevkov and Morata further cements the tight association between Dmp53 and the apoptotic cascade by showing that Dmp53 and JNK mutually activate each other and act upstream of the pro-apoptotic genes  and establish a positive feedback loop that amplifies the initial apoptotic stimuli [An interesting observation is that DΔNp53 induces mitogen expression and proliferation even when apoptosis is inhibited in  mutants , 36, 41. mutants , 36. How stimuli . Dmp53 a stimuli –50. Thus stimuli . Based orpr, hid and grim expression, inhibits DIAP1 and ultimately activates the initiator (dronc) and effector caspases  [Another interesting conundrum is why the AiP program is only induced in response to cell injury, but not in developmental apoptosis. In several fly developmental processes, apoptosis is required to reduce the primordial germ cell numbers in the embryo and eliminate the excess neuroblasts in the larva or the supernumerary interommatidial retinal cell during pupal development, and some of these processes are known to depend on Dmp53 –54. Likee, dcp1) , yet sucDrosophila imaginal wing disc upon growth challenge. Specifically, the authors hampered growth in the posterior compartment of the wing disc, either by the expression of Ricin, a ribosome-inactivating toxin, or by the inhibition of insulin pathway. The cell division and growth rate in the unaffected compartment accordingly slow down to generate an adult wing that is normally proportioned, even though the final size is smaller than the wild-type wing , and reducing or removing Dmp53 drastically diminishes such Hippo phosphorylation, and consequently dampens Hippo pathway activation in the presence a cell-death signal. This work nicely demonstrates that Hippo acts epistatically to Dmp53 to control the activation of apoptosis. Now in the light of the finding that Dmp53 can coordinate organ size and systemic growth in a stress context, an intriguing question begs the answer: can Dmp53 act in concert with the components of the Hippo pathway to control and coordinate tissue growth? And vice versa, does the Hippo pathway play a role in AiP in the classic IR-induced damage model? We do not yet have answers. However, the analysis of yorkie function may hold some clues. It has been well-established that Yorkie activates Diap1 to limit apoptosis during tissue proliferation and growth [Drosophila ASPP1 homologue and Dmp53, which can activate the downstream apoptotic genes [The Hippo signaling cascade is one of the most conserved organ size control pathways in evolution –66. By pd growth . Recentld growth . Reducind growth , 68. Howic genes , 71. TheDrosophila is a genetically tractable model to explore the “primordial” functions of p53 in addition to its well-known functions in mediating apoptosis in the presence of genotoxic stress. In fact, even such canonical Dmp53 function is more complex than we first realized, as studies begin to unravel the roles of Dmp53 isoforms in controlling AiP and programmed cell death at the same time in the same tissue. The molecular details of these processes require future investigation. Furthermore, studies in Drosophila pioneered many founding works in identifying the physiological functions of p53, including the recent discoveries of the role of Dmp53 in controlling organ size, system growth and cell competition in the presence of stress. From an evolutionary point of view, is this kind of growth-control function part of the “primordial” p53 functions? Four years ago, a study from John Abram’s laboratory discovered that Dmp53 mediates meiotic recombination in the germ-line, and proposed that such primordial function of p53 is the origin of the genotoxic stress response [Studies of p53 in the past 35 years have depicted the many facets of p53 functions that are forever evolving. We now recognize that besides being the classic “guardian of the genome” , p53 parresponse . Now an


P53 is most well-known for its tumor suppressive function in differentiated cells. Its activities in embryonic stem cells (ESCs) are, however, less well understood. For many years it was thought that p53 is not active at all in ESCs and unable to elicit a DNA damage response in this cell type. In the last few years, it emerged that p53 may have some functions in ESCs. Nevertheless, it remained a mystery how its activity is controlled in ESCs. A recent report demonstrates that p53 activity is regulated by a novel RNA-containing negative feedback loop that promotes apoptosis specifically in ESCs. This study not only demonstrates unequivocally that p53 is active in ESCs, it further illustrates a novel mechanism of gene regulation–by protein coding RNAs. Cell Stem Cell has shed light on the intricate pathway that regulates the pro-apoptotic function of p53 in ESCs [Embryonic stem cells (ESCs) are pluripotent and can differentiate into many cell types. To avoid passing mutations to daughter cells, ESCs have great need to maintain their genome integrity. The tumor suppressor p53 plays proverbial roles in regulating the genomic integrity of ESCs. Several studies have established that p53 induces ESC differentiation in response to DNA damage , 2. Howe in ESCs .Apela for further study that is repressed by p53 and highly expressed in ESCs. They found that Apela positively regulates p53-mediated DIA in mouse ESCs. Surprisingly, although Apela RNA has a putative coding region, this region is dispensable for the function of Apela in the DIA of mouse ESCs. Instead, Apela acts as a regulatory RNA. But as no direct interaction could be found for Apela and p53, how can this RNA regulate p53 function? To answer this question, the authors performed RNA-precipitation. One of the proteins that precipitated with Apela was the heterogenous nuclear ribonucleoprotein L (hnRNPL). This protein associates with p53 and inactivates the tumor suppressor protein by sequestering it from the mitochondrial department and by stimulating its degradation, the former but not the latter function is antagonized by Apela. In response to DNA damage, the relative interaction between hnRNPL and p53 is decreased, freeing p53 from its constraint and allowing its accumulation and initiation of apoptosis in ESCs. Huang and his colleagues speculate that Apela may facilitate a quick response of ESCs to DNA damage by skipping the translation step. As Apela is repressed by p53, p53, Apela and hnRNPL form a tri-element negative feedback loop in ESCs. Given the enrichment of Apela in ESCs, this negative feedback loop is most likely specific for the regulation of p53 activity in ESCs.To investigate the mechanism, Huang and his colleagues set out to test whether p53 induces apoptosis in mouse ESCs upon DNA damage and found that p53 is critical for DNA damage-induced apoptosis (DIA). Thus, the pro-apoptotic roles of p53 are conserved between stem cells and differentiated cells. They then used genomic approaches and identified a number of potential downstream targets that mediate p53’s pro-apoptotic function. Since little is known about the mechanism of gene repression by p53, the authors decided to focus on one transcript called Because of its highly anti-proliferative activities, p53 needs to be kept in check and released from this constraint when its activity is needed. Therefore, several regulatory mechanisms have been implemented in cells that control p53 abundance and activity in stem cells as well as in differentiated cells including regulation of abundance and activity by posttranslational modifications , associa


It has been shown that p53 has a critical role in the differentiation and functionality of various multipotent progenitor cells. P53 mutations can lead to genome instability and subsequent functional alterations and aberrant transformation of mesenchymal stem cells (MSCs). The significance of p53 in safeguarding our body from developing osteosarcoma (OS) is well recognized. During bone remodeling, p53 has a key role in negatively regulating key factors orchestrating the early stages of osteogenic differentiation of MSCs. Interestingly, changes in the p53 status can compromise bone homeostasis and affect the tumor microenvironment. This review aims to provide a unique opportunity to study the p53 function in MSCs and OS. In the context of loss of function of p53, we provide a model for two sources of OS: MSCs as progenitor cells of osteoblasts and bone tumor microenvironment components. Standing at the bone remodeling point of view, in this review we will first explain the determinant function of p53 in OS development. We will then summarize the role of p53 in monitoring MSC fidelity and in regulating MSC differentiation programs during osteogenesis. Finally, we will discuss the importance of loss of p53 function in tissue microenvironment. We expect that the information provided herein could lead to better understanding and treatment of OS. P53 is a guardian of cell differentiation.P53 regulates genomic stability, growth, proliferation, and immunoproperties of mesenchymal stem cells (MSCs).P53 is a negative regulator of osteogenic differentiation of MSCs.Loss of function of p53 in MSCs compromises their osteogenic differentiation and affects the properties of bone tumor microenvironment (BME) components, therefore it dictates the conditions for osteosarcoma (OS) development.in vivo and in vitro key molecules involved in the process of bone remodeling, in the context of loss of function of p53.To identify Are there any molecules produced by p53-null MSCs that could affect osteoclast properties and compromise bone homeostasis?How do they relate to the diagnosis and prognosis of OS?3 Having been discovered since 1979, p53 is the most studied member of the family with over 60 000 papers so far published. This large mass of scientific data evidentiate a huge complexity for p53 functional program, ranging from the regulation of metabolism6 and mitochondria/oxygen radicals8 to the deeply analyzed DNA damage repair system,14 autophagy,16 and, last but not the least, its role in cell stem maintenance and lineage determination.18 Despite all these investigations, efforts, and advances in knowledge, many crucial intriguing points still remain unanswered to fully understand the physiological and pathological role of p53. These wide range of effects raise from several angles, including, for example, its regulation at the transcriptional level, at the level of micro-RNA,22 and splicing isoforms24 to its translational regulation and its stability/degradation at the protein level.29 In parallel to so much effort in understanding the function of p53, significant efforts are also underway on its potential clinical exploitation.37 Although being identified after ~20 years, already now, p63 and p73 show a similar complexity, and also the ability to interact with p53 at the structural and functional level,49 where the p63 function is highly relevant in skin formation and homeostasis,51 as well as in cancer53 and stem cell regulation.57TP53 belongs to the so-called ‘p53 gene family' of transcription factors, which includes also the proteins p63, p73, and p53 itself.58 Thus, a single gene can exist in multiple isoforms with distinct biological functions.60 P53 protein, encoded by the TP53 gene in humans and the Trp53 gene in mice, is well known for its role as the ‘guardian of the genome' and exerts a pivotal role in maintaining the genetic stability.63 It can prevent tumor formation by regulating cell cycle,64 apoptosis,65 senescence,66 and metabolism67 by binding to responsive elements on DNA (p53RE).68 Abnormal regulation of the p53 family has a critical role in tumorigenesis; indeed, TP53 mutations have been detected in over 50% of all human cancers.70The p53 family of transcription factors have several members including p53, p63, and p73. Each member of this family expresses unique mRNA variants resulting from alternative splicing, promoters, and transcription initiation sites.TP53 and/or the retinoblastoma gene RB1 have been reported to be the main causes of the development of sporadic OS.71In vitro experiments comparing MSCs with malignant OS cells, as well as in vivo studies using transgenic mice with targeting p53 and Rb  silencing in MSCs, have elegantly demonstrated that when p53 alone was deleted, the incidence of OS could reach 60%.72 Another function of p53 in suppressing tumor is to act as ‘a guardian of differentiation'.59Silent mutations in the tumor suppressor gene 74Notably, p53 guards osteogenic, myogenic, adipogenic, hematopoietic, and neural differentiation of adult stem cells.75 Seven subtypes of OS have been characterized according to histological analysis of the osteoid matrix produced by aberrant osteoblasts: osteoblastic, fibroblastic, chondroblastic, telangiectatic, epithelial, small cell, and giant-rich cell.76 The abundant deposition of osteoid matrix and osteoblast-like features of the malignant cells are the dominant characteristics of the osteoblastic phenotype. This subtype manifests the highest incidence representing 75% of screened OS.78 Within OS of the osteoblastic subtype, aberrant preosteoblasts and osteoblasts produce their own osteoid mineralized matrix close to the area of growth plate (GP). Although chromosomal abnormalities have a decisive role in the development of OS,79 the karyotype is not essential for the subtype classification. OS frequently occurs in human patients with Li-Fraumeni syndrome and with hereditary retinoblastoma. Li-Fraumeni patients carry a germline p53 mutation in one allele compromising the function of p53.82 Different studies have reported that preosteoblasts and osteoblasts represent the cells of origin of OS.83 Importantly, cellular microenvironment is also decisive in determining the fate of stem cells and in promoting tumor formation.84 The osteogenic differentiation of p53-deficient or mutant MSCs can be affected by signals from BME and promote eventually OS. Intrabone inoculation of undifferentiated p53−/− and p53−/−Rb−/− MSCs generated osteoblastic OS and developed metastasis characterized by osteoid areas in the lung, spleen, and heart.33 These data suggest that, along with specific bone microenvironment conditions, undifferentiated MSCs with compromised p53 function can represent the cells of origin of OS  released during bone resorption. Insulin-like growth factors and transforming growth factor-beta (TGF-β) are examples of two coupling growth factors secreted during bone degradation, which have been proven to stimulate the osteoblast activity.88 However, MSCs and osteoblasts can also secrete molecules that can influence the osteoclast activity, and affect bone remodeling in the same cases.89 Indeed, throughout the lifetime bone remodeling is kept at a constant rate to balance bone formation and bone degradation, and to guarantee bone homeostasis. However, along with aging, this exquisite equilibrium is subjected to alterations mainly as a consequence of hormonal dysfunctions. Indeed, steroid hormone deficit enhances the resorbing activity of osteoclasts, which could terminate in an osteoporotic condition.90 Conversely, an increase in bone mass and bone density is representative of osteosclerosis and osteopetrosis conditions. Higher intake of bone in osteosclerosis is exclusively promoted by osteoblasts, whereas osteopetrosis is due to aberrant activity of osteoclast-mediated bone resorption.91 Notably, during the process of endochondral bone formation, which occurs until adolescence, the mesenchymal progenitor cells first differentiate into chondrocytes, which generate new cartilage on the GP. The chondrocytes will be slowly replaced by osteogenic progenitor cells and osteoblasts to produce the bone.84 Interestingly, under normal conditions p53 acts as a negative regulator of osteoblastogenesis by repressing the promoter activity of transcription factors required in the early phase of osteogenic commitment, such as Osterix,92 Cbfα-1, and Runx2 in osteoprogenitor cells93 .101 P53 can regulate key transcription factor genes involved in exclusive programs of differentiation and dedifferentiation of somatic cells, with an impact on stemness and development. Given that p53 is a tumor suppressor gene and gate keeper of cell differentiation, in this review we want bring to light the connection of p53 status in MSCs, BME, and OS development. Notably, p53-null MSCs exhibit accelerated growth rate and aberrant osteogenic differentiation compared with wild-type MSCs, which contributes to tumor bone formation. Indeed, distinct studies highlight that p53-deficient bone marrow-derived MSCs can proliferate faster, and appear to differentiate earlier during in vitro osteogenic differentiation compared with the wild-type MSCs.102 However, this ‘tricky' appearance to differentiate earlier into osteoblasts reflects a more complex scenario; indeed, p53-null MSCs are impaired in achieving terminal differentiation towards mature osteocytes.92 MSCs represent a source of precursor for osteogenic progenitor cells and osteoblasts. P53 mutations that lead to defects in the control of cell growth of osteogenic progenitor cells represent the main source of sporadic OS.MSCs are a subset of adult progenitor cells that exist in almost all adult tissues . Adult MSCs have proven to be cells of mesodermal origin, which can give rise to skeleton, muscle, heart, spleen, and other internal organs.in vitro knockdown of p53 in mouse embryonic fibroblasts (MEFs), which are cells representative of an embryonic stage of development, induced higher expression level of Osterix and Runx273 but not of terminal differentiation markers such as Osteocalcin and Sost-Sclerostin.103 Conversely, p53 knockdown in multipotent bone marrow stromal cells (MBA-15), which resembles adult progenitor cells,104 promoted terminal osteogenic differentiation.73 Consistently, also after reintroduction of wild-type p53 in OS cell line, apoptosis and terminal differentiation were promoted.104 We can emphasize that p53 can regulate bone formation and the differentiation of early osteogenic precursors as demonstrated by the knockdown of p53 in MEF, and, furthermore, it can also promote terminal differentiation in MBA-15.The in vitro for several passages without being severely compromised in their properties.105 The induction of p21 or cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 mainly promoted by p53 is associated with cell-cycle arrest  and C/EBP-α (CCAAT/enhancer-binding protein-α).114 Adipocytes and osteoblasts are both cells derived from multipotent progenitor cells. The undifferentiated status of the cells is kept by the repression of transcription factors that repress each other to preserve multipotency. However, upon appropriate stimulations, MSCs make sequential cell fate choices.115 The commitment of MSCs towards a specific mesenchymal differentiation program is coordinated also by p53, which regulates their multipotential state. In in vivo studies, p53 is upregulated in adipocytes from genetically obese mice in a fed state. Transgenic mice overexpressing functional p53 gain less body mass and adipose tissue when compared with wild-type mice; this has suggested an inhibitory role for p53, which may be exerted by changes in metabolism.116 Indeed, in the absence of functioning p53, a shift from oxidative phosphorylation towards glycolysis was observed.117 Furthermore, even myogenic differentiation can be monitored by p53. Distinct studies have shown an increase in p53 mRNA levels during myogenic differentiation in vitro.120 P53 might be involved in this process by regulating the retinoblastoma protein, Rb, which has a pivotal role in the differentiation of muscle through cell-cycle arrest and also by specific genes involved in the myogenic differentiation program.123 The association of p53 expression and cell-cycle regulators, which are target genes of p53, such as p21, was observed during the development of the mouse nervous system.125 Indeed, p53 monitors the differentiation of neural stem cells via its regulating pathways including cooperation with phosphatase and tensin homolog (PTEN).125 Notably, accumulation of mutant p53 in neural stem cells in the subventricular zone of the brain could generate aberrant neural progenitor cells and promote glioma formation.126 However, it is still unclear how p53 functions in the specific signaling context to regulate neural stem cells differentiation.127 In hematopoiesis, p53 induces hematopoietic stem cells to differentiate into proper mature blood cells and function in maintaining their quiescence.73 So far, the development of skeletal muscles and blood system has not yet been found abnormal in p53-null mice. Several in vitro studies have confirmed the role of p53 as a negative regulator in cell differentiation pathways, which reflects the complexity of the underlying mechanisms , necessary for metabolism adaptation, avoiding its proteasomic degradation through murine double minute 2 protein.134 Ablation of p53 increases the expression of HIF-1-α in a hypoxia condition, which in turn induces the expression of vascular endothelial growth factor (VEGF) in tumor cells and promotes angiogenesis, neovascularization, tumor growth, and invasion. VEGF and HIF-1-α are, furthermore, overexpressed in several types of human cancers, especially with HIF-1-α in metastatic OS.135Interestingly, our previous work has proved that MSCs that lack p53 exhibit tumor-promoting characteristic through high secretion of nitric oxide (NO) and higher vigorous immunomodulation when compared with wild-type MSCs.84 Given the plasticity of MSCs to generate and differentiate into several cell types, including osteoblasts, it is not surprising that MSCs with p53 aberrations have been suggested to be the cells of origin for bone tumor, including OS, chondrosarcoma, Ewing's sarcoma, and sarcoma.75 We previously have discussed about the impaired osteogenic differentiation of p53-deficient MSCs; however, we here want to emphasize that p53-null MSCs also represent an important cellular component of tumor microenvironment. Interestingly, it has been demonstrated that in tumor BME, MSCs can support OS growth through the expression of CCL5 (chemokine ligand 5).136 Interestingly, CCL5/CCR5 (C–C chemokine receptor type 5) axis can promote OS migration through the extracellular signal-regulated kinase pathway, which induces the nuclear factor κ-light-chain enhancer of activated B cells (NF-κb).136 Conversely, growth factors secreted from cancer cells and during bone resorption, such as tumor necrosis factor-α, TGF-β, bone morphogenetic protein 2 (BMP2), and interleukin-6 (IL-6), can promote osteoclast maturation by directing the expression of RANK (receptor activator of the receptor activator of nuclear factor-κb) on the surface of osteoclast precursor cells increasing bone erosion. The increased bone degradation culminates in the release of BMP2 and TGF-β, which severely contribute to tumor growth and stimulate the osteoclast activity.137 TGF-β can evoke MSC-secreted IL-6, which acts by promoting OS metastasis via STAT-3 .138 These data emphasize how the loss of function of p53 is a determinant in dictating the conditions that contribute to initiate OS: on the one hand, it can affect proliferation, immunoproperties, and compromise osteogenesis of undifferentiated MSCs, but on the other hand, it can affect the properties of BME components compromising the talk between BME and cancer cells, a further condition that supports OS initiation and development.During bone growth, several factors and extracellular matrix components secreted by mesenchymal progenitor cells and chondrocytes in the GP will recruit BME components to guarantee a balanced bone remodeling. The BME hosts different types of cells, including osteoblasts, osteoclasts, mesenchymal cell precursors, hematopoietic stem cells, chondrocytes, and endothelial cells, as well as fibroblasts stromal cells and immune cells. In this special scenario, p53 status has a determinant role . In the Wang et al.92 and other groups have proposed a negative role of p53 in regulating osteogenesis and other mesenchymal differentiation programs. We set the evidence that p53 mutational events occurring in undifferentiated MSCs or in osteoblasts at different stages of commitment can promote OS initiation72 as a consequence of alterations of osteogenic differentiation, bone remodeling, and bone homeostasis.139 Indeed, OS is a heterogeneous tumor that includes cells at different stages of commitment during osteogenesis.140 Interestingly, the osteosclerotic condition observed in p53-null mice imposes the phenotype of OS development.106 We summarize that p53 can affect osteogenic differentiation of MSCs and largely contribute to OS initiation: (1) it can promote or abrogate differentiation of multipotent progenitor cells acting as a negative mediator of transcription factors of early osteogenic differentiation; (2) it can regulate the genomic stability, growth, and proliferation of MSCs; (3) it can affect the immunoproperties of MSCs through growth factors and chemokine secretion; (4) it can affect the BME-regulating immune properties, growth, proliferation, and differentiation of microenvironment components. Further investigations on the molecular mechanisms through which loss of function of p53 can affect properties of MSCs and osteoprogenitor cells should be considered. This will ameliorate the knowledge of p53 function in the context of bone biology, and also will be helpful in identifying new strategies for targeting key molecules necessary for OS formation and survival.In this review, we aimed to bring to light that p53 has a pivotal role in keeping the balance between bone formation and bone degradation. Indeed, p53 not only regulates the genomic stability of MSCs but also their osteogenic differentiation functioning as ‘bone remodeling surveillant' to prevent bone tumor initiation.


Upon activation, p53 leads to cell-cycle arrest and promotes DNA repair or induces apoptosis via several pathways. p63 and p73 are structural homologs of p53 that can act similarly to the protein and also hold functions distinct from p53. Today more than 40 different isoforms of the p53 family members are known. They result from transcription via different promoters and alternative splicing. Some isoforms have carcinogenic properties and mediate resistance to chemotherapy. Therefore, expression patterns of the p53 family genes can offer prognostic information in several malignant tumors. Furthermore, the p53 family constitutes a potential target for cancer therapy. Small molecules  have been objects of intense research interest in recent years. They restore pro-apoptotic wild-type p53 function and were shown to break chemotherapeutic resistance. Due to p53 family interactions small molecules also influence p63 and p73 activity. Thus, the members of the p53 family are key players in the cellular stress response in cancer and are expected to grow in importance as therapeutic targets. Human cells are constantly exposed to external and internal stressors, which cause damage to the integrity of the cell and to its genome. In order to guarantee the survival of the organism, cells have developed numerous strategies to adapt to stressors. In this review, we would like to discuss the influence of cellular stress on tumor development as well as strategies in cancer therapy targeting pathways involved in cell-cycle control and apoptosis. Special emphasis is put on the members of the p53 family.The development of cancer is a multistep process that involves a series of mutations in the progenitor cell . It enabWhile p53 has been known for more than three decades, two further members of the p53 family, p63 and p73, have been discovered more recently. The three genes exhibit a high degree of homology and there is increasing evidence that they have risen from the triplication of a common ancestral gene , 25. AllWhile p63 is crucially involved in craniofacial, limb, and skin development , p73 plaMalignant tumors often exhibit defects in apoptosis signaling pathways, resulting in tumor cell survival. Therefore, understanding the exact mechanisms of apoptosis can provide new strategies for the development of anti-cancer treatments. The extrinsic apoptosis signaling pathway is initiated by ligands such as TNFα, CD95L, and TRAIL binding to death receptors –38. The c from the mitochondrial intermembrane space  (core domain) is shared by most p53 isoforms and binds to response elements of target genes. A large number of p53 mutations occur within this region of the gene . The N-tp53 controls a large number of genes mediating G2/M and G1 cell-cycle arrest, DNA damage recognition, DNA repair, apoptosis, and senescence  Figure , 56. EveAnother important upstream regulator of p53 activity is p14ARF, a protein transcribed from an alternate reading frame of the CDKN2A gene locus that also encodes for the tumor suppressor p16INK4a , 68. p14c release , which plays an important role during neurogenesis. TAp73 knock-out mice show reduced levels of p75NTR and suffer from peripheral nerve defect, including myelin thickness and thermal sensitivity , which is essential for the oxidative pentose phosphate pathway . Cox4il p73 is rarely mutated in human cancer (<1%), but overexpression of p73 can be found in several malignancies, for example, in hepatocellular carcinoma , 162, neLarge-scale genome sequencing has shown that over half of human malignancies exhibit point mutations in the p53 gene impairing p53 function. Most p53 mutations are missense point mutations located within the DBD. Many of them lead to destabilization of folding of the domain at physiological temperatures and interfere with its DNA-binding ability . Certainin vitro and in vivo settings . RITA binds p53 and thereby induces conformational changes within the molecule that prevent MDM2 association , 196. In in vivo . The ant in vivo .Rational design led to construction of the spiro-oxindole MI-219, which is a highly specific small molecule inhibitor of p53/HDM2-interaction . Later, As an alternative to interfering with p53/MDM2-interaction, degradation of p53 can be prevented by inhibiting the E3 ligase activity of MDM2, and therefore, preventing ubiquitination of p53 . A serieThe tryptamine JNJ-26854165 (Serdemetan) effectively prevents p53/HDM2 from binding to the proteasome, thereby inhibiting degradation of p53 . In acutSIRT1, a nicotinamide adenine dinucleotide-dependent class III histone deacetylase, deacetylates p53 at Lys382, thereby reducing its activity . Hence, MET (APR-246), a compound that bears great structural similarities to PRIMA-1, but has higher activity than its predecessor, was discovered  in advanced prostate cancer and hematological malignancies, as well as a phase Ib/II clinical trial using this compound in addition to carboplatin in recurrent high-grade serous ovarian cancer are under way and will offer more insight into the effectiveness and practicability of mutant p53 reactivation .PRIMA-1 in vivo , 225. Alin vitro and in a mouse model (MIRA-1 (mutant p53 reactivation and induction of rapid apoptosis) is a maleimide-derived molecule and has no structural similarity with PRIMA-1, but it is equally able to restore p53 function leading to cell death via apoptosis with even higher potency than PRIMA-1 . By reesse model . To dateAlthough PRIMA-1 and MIRA-1 seem to have stabilizing effect on a great variety of p53 mutants, they are not able to restore normal protein configuration to the Phe176 mutant . This shCP-31398 was discovered by screening a library of more than 100,000 synthetic compounds for substances that effectively stabilize p53 conformation . InitialIn vivo, in a xenograft mouse model, tumor growth could be decelerated by intraperitoneal injection of RETRA (However, increased p53 activity bares risks for non-cancerous cells that might also be subject to apoptosis and further research is needed to find the adequate dose-response relationship, specific to the compound used . In an aof RETRA . AlthougMoreover, restoring p53 apoptotic function and modulation of p63 and p73 expression is often essential for sensitivity toward chemotherapeutic drugs or radiation, as lack of p53 and unfavorable expression patterns of p63 and p73 can lead to resistance toward treatment in different malignant tumors –235. RecHowever, restoration of wild-type p53 might not be beneficial in all types of tumors. Jackson et al. showed that doxorubicin lead to cell-cycle arrest and senescence instead of cell death in breast cancer expressing wild-type p53, thereby promoting tumor cell survival and resistance to chemotherapy . This shThe p53 family plays a central role in cancer development and treatment response. Whereas p53 is often mutated in tumors, p63 and p73 function is preserved, yet altered by different expression patterns of their TA and ΔN isoforms. Increasingly, these expression patterns are evaluated to estimate prognosis and adapt anti-cancer therapy. Nevertheless, the molecular mechanisms regulating the interplay between the different isoforms of the p53 family are only partly understood and are focus of current research. Identifying compounds that interfere with oncogenic signaling induced by certain p63 and p73 isoforms could be a novel approach in anti-cancer therapy.An increasing number of compounds that re-establish pro-apoptotic p53 function in cancer cells have emerged over the past decade. A variety of small molecules, which aim at increasing p53 function in cancers expressing wild-type p53, have been discovered. Among them are Nutlins, which are already undergoing clinical evaluation, RITA, tenovins, and many others.In tumors with underlying p53 mutation restoring wild-type activity of p53 has proven more difficult, but nevertheless feasible. PRIMA-1 and MIRA-1 are effective at inducing apoptosis via p53 in tumors that exhibit a great variety of p53 mutations. Yet, there are other small molecules, like PhiKan083, which are more specific and restore wild-type configuration of specific mutants only.in vivo studies and clinical trials have shown synergistic effects of small molecule treatment and chemotherapeutic drugs in a variety of malignancies. Especially cancer cells, which are resistant to chemotherapy due to impaired p53 function, become more susceptible to treatment.A number of Taking the approaches of p53 reactivation further, there might be new possibilities of targeting CSCs, which are often insusceptible to chemotherapy. Induction of p53 in these cells could lead to activation of pro-apoptotic pathways via differentiation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The standard classification used to define the various cancer genes confines tumor protein p53 (TP53) to the role of a tumor suppressor gene. However, it is now an indisputable fact that many p53 mutants act as oncogenic proteins. This statement is based on multiple arguments including the mutation signature of the TP53 gene in human cancer, the various gains-of-function (GOFs) of the different p53 mutants and the heterogeneous phenotypes developed by knock-in mouse strains modeling several human TP53 mutations. In this review, we will shatter the classical and traditional image of tumor protein p53 (TP53) as a tumor suppressor gene by emphasizing its multiple oncogenic properties that make it a potential therapeutic target that should not be underestimated. Analysis of the data generated by the various cancer genome projects highlights the high frequency of TP53 mutations and reveals that several p53 hotspot mutants are the most common oncoprotein variants expressed in several types of tumors. The use of Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, such as ‘amorph', ‘hypomorph', ‘hypermorph' ‘neomorph' or ‘antimorph', allows a more meaningful assessment of the consequences of cancer gene modifications, their potential clinical significance, and clearly demonstrates that the TP53 gene is an atypical cancer gene. However, their relatively infrequent mutation and/or association with specific tumor groups could limit their value as biomarkers or therapeutic targets.6The development of next-generation DNA sequencing methods has led to a burst of information, including the release of the sequences of >5000 cancer genomes, an achievement inconceivable only 15 years ago.7 Therefore, drug discovery and development programs in academia and industry have mostly focused on kinase inhibitors and other approaches designed to inhibit activated oncoproteins, whereas relatively few therapeutic strategies to reactivate tumor suppressors have been developed to date.In general, cancer genome studies highlight oncogenes as the most promising targets for drug development mainly because these hyperactive protein variants appear to be more easily druggable than the products of tumor suppressor genes, inactivated by heterogeneous nonsense or frameshift mutations.et al.8 in the 1960s, showing that fusion of normal cells with tumor cells resulted in a normal phenotype. Loss of specific chromosomes from the hybrid cells led to reappearance of the tumor phenotype, suggesting the existence of critical genes able to suppress the tumor phenotype. The tumor suppressor gene concept was also supported by Knudson's epidemiological studies on familial and sporadic retinoblastoma and the subsequent loss of heterozygosity (LOH) analysis that identified chromosomal deletions in tumors.10 Haber and Harlow defined tumor suppressor genes as ‘genes that sustain loss-of-function (LOF) mutations in the development of cancer'.11 This classification has evolved over time and novel classes such as caretakers, gatekeepers or landscapers have been added to take into account the function of novel genes and their association with tumor development.12 Although oncogene and tumor suppressor gene alterations may have direct consequences on cell growth, caretakers and landscapers act indirectly by promoting either genetic instability or an abnormal cellular environment that will foster neoplastic transformation. This classification, based on the diversity of the functional activity of the wild-type product, should not be confused with the consequences of their alteration.The first evidence for the existence of tumor suppressor genes came from cell fusion experiments performed by Harris 14 Although the two-box oncogene/tumor suppressor categorization remains a central concept in tumor biology, it is becoming increasingly obvious that it is difficult to put some genes into one box. As cancer research develops rapidly, it is essential to ensure highly flexible data classification so that labels do not paralyze the emergence of new concepts.For example, LOF is still the main characteristic of tumor suppressor genes, but the Haber and Harlow definition does not take into account the heterogeneity of the different protein variants or other activities such as dominant negative (DN) effects on wild-type protein or gain-of-function (GOF). Furthermore, for some genes such as Notch, this definition can be cell-type specific, as the spectrum of mutations is different in blood and solid tumors, with different consequences for the protein.versus inactivated, despite accumulating evidence that the majority of mutant p53 proteins are heterogeneous oncogenic proteins with multiple GOF activities and with potential as therapeutic targets.The status of TP53 as a tumor suppressor gene can be used as a paradigm. Wild-type TP53 clearly acts as a negative regulator of cell growth, but considering TP53 mutations solely as LOF mutations would prevent a full understanding of how TP53 mutations drive tumor growth, as TP53 status in human cancer is often defined in binary terms, wild-type In this review, we will show that, despite the important diversity of TP53 mutations, some specific p53 mutants are among the most frequent variants expressed in human cancer. In addition, we have gathered various lines of evidence accumulated over 35 years of research that illustrate how TP53 is an out-of-the-box entity. Finally, using Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, we will discuss how this classification would be more appropriate to define the heterogeneous effects of cancer gene mutations.18 More than 80% of somatic and germline TP53 alterations are missense mutations that lead to the synthesis of a stable mutant protein that accumulates in the nucleus of tumor cells . Although their method can separate classical oncogenes from tumor suppressor genes in general, the TP53 gene would still straddle the boundary between the two classes of genes.Oncogenes are typically activated by missense mutations that target specific key residues of the protein . Tumor s19The strong selection to maintain expression of full-length p53 protein in tumors is highly suggestive of a vital role in transformation, including DN activity and/or GOF. As >90% of TP53 mutations are localized in the core domain of the protein (residue 100 to 300), all p53 protein isoforms are affected by these alterations.20 This vast scattering of TP53 mutations is due to the marked fragility of the core domain, which can be destabilized by amino acid substitutions at many different positions.21The distribution of mutations in the p53 protein is also unique among all cancer genes, including oncogenes and tumor suppressor genes. All but 7 residues of the 393 amino acid residue p53 protein have been the target of at least 1 mutation in human cancer, and in the core domain that contains the DNA-binding region, each residue has been found to be mutated at least 5 times in independent tumors, and up to >2,000 times for hotspot mutants.22 The data include genomic, epigenomic, transcriptional and proteomic information. Not surprisingly, TP53 was found to be the most frequently mutated gene in this new set of data  and homozygous  animals. This is an important finding because it rules out the possibility that the inability of mutant p53 to act as a transcription factor and induce sufficient levels of MDM2 protein, a p53-specific E3 ubiquitin ligase that targets p53 for modification and subsequent degradation by the 26 S proteasome, is a cause for mutant p53 hyperstability.Terzian 30 The CHIP E3 ligase participates in p53 degradation by forming a large complex that includes HSP90 and HSP70. Mutant p53 forms stable complexes with both HSP, CHIP and MDM2, inhibiting the ligase activity of both. A large survey of tumor cell lines expressing wild-type and mutant p53 showed that stabilization of endogenous mutant protein is due to a complete lack of ubiquitination.31 Inhibition of HSP90, either via knockdown or by specific drugs, alleviates the formation of such complexes, leading to reactivation of both MDM2 and CHIP, and mutant p53 degradation.However, additional ubiquitin ligases have recently been identified that participate in the degradation of the tumor suppressor, including Pirh2 (p53-induced protein with a RING-H2 domain), Trim24 , COP1 (constitutive photomorphogenesis protein 1) and CHIP (C-terminus of Hsc70 interacting protein).35 Nuclear magnetic resonance, circular dichroism and X-ray diffraction analyses have confirmed and expanded this description and suggested that multiple thermodynamic stages are associated with the various mutations depending on their position.38 This heterogeneity extends to the biological activity of mutant p53 proteins.39 Analysis of the transcriptional activity of 2500 p53 mutants on 8 different target genes representative of different TP53 functions demonstrated heterogeneous penetrance of the various p53 mutants with >50% presenting only partial loss of activity.43The heterogeneity of p53 mutants was discovered 25 years ago with the description of the so-called ‘structural' mutants, in which the DNA-binding domain is unfolded (e.g. p.R175H), and ‘DNA contact' mutants, in which residues interacting directly with DNA are substituted .in vitro and in vivo systems . Further studies have largely supported this observation. Mutant p53 GOF includes enhanced tumorigenesis, metastasis, resistance to therapy and genomic instability. The genomic instability effect was further supported in a recent analysis of 3000 tumors showing that TP53 mutations were strongly associated with a high frequency of copy number changes.49 GOF in p53 mutants is highly heterogeneous, an observation that may be related to the various conformations of p53 mutants. GOF mechanisms may be the result of changes in the specificity of the DNA-binding activity of the p53 mutant, leading to the induction of novel transcriptional programs, or changes of its interaction with other cellular proteins directly or indirectly related to the regulation of gene expression. Several transcriptional programs are specifically activated by p53 mutants, most of them resulting in increase of tumorigenicity but whether this is mediated by a direct binding of the mutated protein to a specific DNA sequence or via an interaction with other transcription factors remains to be elucidated52 , an observation adding one more grain to the important relation between TP53 and metabolism. This observation is also in line with the study by Zhang et al.55 showing that p53 mutants stimulates the Warburg effect in cancer cells.Mutant p53 GOF has been extensively investigated in multiple 57 TP63 and TP73 are both expressed as many isoforms due to alternative usage of promoters for transcription and alternative splicing. Long isoforms (p73 or p63 containing the transactivation domain (TA-p73 or TA-p63)) are able to transactivate the same target genes as p53 and induce apoptosis, while short forms (amino-deleted p63 or p73 isoforms (DN-p63 or DN-73)) have an opposite activity via DN mechanisms. TP63 and TP73 are able to cooperate with TP53 to induce apoptosis, suggesting the existence of a complex network of interactions between the products of these three genes. p53 mutants with unfolded structure, but not DNA contact mutants, bind specifically to p63 and p73 and impair their apoptotic activity  increased the complexity of this network, as the two p53 homologs might also contribute to its oncogenic potential.An important issue that has never been fully analyzed is the consequence of accumulation of mutant p53 protein in tumor cell nuclei. Studies have shown that this accumulation may be as much as 10-100-fold higher than that of wild-type protein, which raises questions concerning the specificity of some mutant p53 activities and how some nonspecific squelching effects may be associated or confounded with these novel properties. Furthermore, this aspect also raises the issue of tumor heterogeneity, as p53 accumulation in human tumors is highly heterogeneous, even for single p53 variants, and could be associated with the genetic background of the tumor and the individual.63 Cells from mice deficient for the three major TP53 target genes, CDKN1A (the gene encoding p21), BBC3 (the gene encoding PUMA) and PMAIP1 (the gene encoding Noxa) (p21−/−puma−/−noxa−/− mice) are deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence, but these animals also remain tumor-free.64 Taken together, these observations suggest that TP53 driver mutations are selected to impair specific TP53 pathways that remain to be identified, and only mutants defective in such transcriptional programs will be selected in human tumors.65The general idea that loss of transcription in mutant p53 is the driving force selected during tumorigenesis also needs to be reevaluated, as it is far from straightforward. Surprisingly, mice expressing p53 mutants transcriptionally defective for the three canonical pathways, growth arrest, senescence and apoptosis, are not prone to cancer.66 Homozygous TP53−/− mice are highly prone to cancer, in particular T-cell lymphoma and sarcoma. Although these knockout mice supported the model of TP53 as a tumor suppressor gene, they were not fully satisfactory, since most cancers in humans are carcinomas, which were observed at very low frequency in TP53 knockout mice.68 Improvements in the production of transgenic mice led to the creation of knock-in mice expressing various hotspot mutants that include structural or DNA contact mutants and result in more ‘human-like' tumors.70 Compared with TP53−/− mice, these novel knock-in models displayed a higher degree of heterogeneity in the spectrum of tumor types with more frequent carcinomas. Furthermore, these tumors were highly invasive and metastatic, a feature absent in TP53−/− mice. Two characteristics of these mice models support the notion of heterogeneous GOF for p53 mutants. First, the spectrum of tumors differed according to the various mutant alleles used in these mice. For example, the ‘DNA contact' mutant p.R270H (p.R273H in humans) results in a high frequency of carcinomas, whereas the ‘structural' mutant p.R172H (p.R175H in humans) predominantly results in osteosarcomas. Second, the tumor spectra of heterozygous TP53 p.R172H/− or TP53 p.R270H/− mice were different from those of TP53+/− or TP53−/− mice, which argues against any differences due to a DN activity toward wild-type p53. Some p53 mutants such as p.R175P are defective in activating genes associated with apoptosis without impairing growth arrest.71 Onset of tumors in knock-in mice expressing this mutant (p.R172P in mice) is delayed compared with TP53−/− mice or mice expressing hotspot mutations.72 In contrast to tumors expressing other p53 mutants, these tumors were mostly diploid, indicating that the growth arrest function of TP53 could be essential for maintaining genetic stability, but is not the primary tumor suppressor function of wild-type TP53.The first TP53 knockout mice were generated by removing exons encoding the DNA-binding domain, thus impairing the expression of all p53 isoforms.74 This is a major challenge, for several reasons. Mutant p53 is clearly a different kind of target compared with those of successful novel anticancer agents such as Herceptin (trastuzumab) and Gleevec (imatinib) that block critical oncogenic kinases overexpressed in various tumors. In the case of mutant p53, the main aim is to refold and reactivate a dysfunctional tumor suppressor  in cells.NSC319726 (ZMC1) is another mutant-specific p53 reactivator. Carpizo and co-workers81 PRIMA-1 and its structural analog PRIMA-1Met (APR-246) refold mutant p53, enhance expression of several TP53 targets, including Bax, Puma and Noxa, and inhibit human tumor xenografts in SCID mice and mouse tumors in syngeneic hosts. These effects are observed with a range of mutant p53 proteins. APR-246 synergizes with several chemotherapeutic drugs, for example, adriamycin and cisplatin, to induce mutant p53-dependent tumor cell death.82 Both PRIMA-1 and APR-246 are converted to the Michael acceptor methylene quinuclidinone (MQ) that binds covalently to cysteines in the p53 core domain83 MQ binding to p53 is sufficient for mutant p53 reactivation, as shown by protein transfer experiments with APR-246/MQ-treated recombinant p53. It is currently not clear to which p53 cysteines MQ binds, although docking simulations and functional studies in cells have indicated that C124 is one possible target.84 APR-246 has been tested in a phase I/II clinical study in patients with hematological malignancies or hormone-refractory prostate cancer.85 This study showed that APR-246 is safe and has a favorable pharmacokinetic profile. Biological effects consistent with p53 reactivation were observed in patient leukemic cells after treatment, and clinical responses were observed in two patients, including one AML patient with a p53 p.V173M core domain mutation.The small molecules PRIMA-1 (p53 reactivation and induction of massive apoptosis-1) and MIRA-1 (mutant p53-dependent induction of rapid apoptosis-1) were identified in a cell-based screen of the NCI Diversity set.86 thereby increasing cellular ROS levels. Moreover, Tessoulin et al.87 found that APR-246 can decrease GSH levels and thus impair the redox balance in multiple myeloma cells independently of p53.In addition to targeting p53, APR-246 has been shown to inhibit thioredoxin reductase (TrxR1) and convert the enzyme to an NADPH oxidase,74 Their mechanisms of action are not fully understood and they have not yet been tested in the clinic.Various other small molecule reactivators of mutant p53 have also been identified, including CP31398, Ellipticine, P53R2, SCH529074 and stictic acid reviewed in the study by Bykov and Wiman.88 Disruption of complexes between mutant TP53 and TP63/p73 by RETRA leads to restoration of expression of TP53 target genes and tumor suppression.In light of the growing evidence in favor of GOF activities of mutant p53, a more modest but still attractive aim for therapeutic targeting of mutant p53 is inhibition of the oncogenic properties of mutant p53 . This ma19 . Restoration of nonsense TP53 mutations will obviously require different approaches than those described above for missense mutations. Interestingly, aminoglycosides, for example, gentamicin and G418, have been shown to induce read-through of the c.637C>T (p.R213*) mutant p53 and expression of full-length p53.89 Although clinical use of these drugs is limited by their toxicity, the results nonetheless demonstrate that induction of read-through of premature stop codons in nonsense mutant p53 is feasible and suggest that high throughput screening for more efficient and less toxic read-through-inducing compounds should be carried out. Questions remain as to the activity of the full-length p53 protein induced on translational read-through and to what extent pharmacological induction of read-through of premature stop codons will induce read-through of natural stop codons. If these problems can be solved, induction of read-through could be a useful strategy not only for reactivation of nonsense mutant p53, but also for reactivation of other tumor suppressors that are frequently inactivated by nonsense mutations, for example APC and PTEN (phosphatase and tensin homolog gene).A significant fraction (8%) of TP53 mutations are nonsense mutations that give rise to expression of a truncated and inactive p53 protein. The c.637C>T (p.R213*) mutant is the most common nonsense mutation in TP53, and is actually more common than many missense mutations in human tumors90 suggested a classification of mutations based on quantitative analysis of wild-type ‘characters'. He proposed the terms ‘amorph, ‘hypomorph', ‘hypermorph', ‘neomorph' or ‘antimorph'. The significance of these terms was subsequently modified, as they were proposed at a time when the relationship between a gene and its product was not clearly established.90 These terms can now be used to classify cancer genes to more clearly understand the consequences of cancer gene alterations. This system has enormous benefits, as it is based on the outcome of the mutation regardless of the initial biological function of the gene. It could greatly facilitate the design of optimal drug development strategies.In Muller91 Other mechanisms, such as promoter methylation, loss of expression via microRNA dysregulation or frameshift and nonsense mutations, can also lead to total LOF, but in many cases it is difficult to exclude residual activities that will lead to heterogeneous hypomorphic variants, and for many genes it is likely that true amorphic variants are not as frequent as hypomorphic variants  mutation can be applied mainly to tumor suppressor genes, whose function(s) must be totally impaired to drive tumorigenesis, whereas a hypomorph mutation only leads to partial reduction of activity. From a genetic point of view, true amorphic alterations can be easily associated with genes that sustain biallelic deletions such as PTEN or retinoblastoma gene (RB1).variants . For the93 have been defined as disruption of the wild-type activity by the mutant polypeptide  or the potassium channel tetramerization domain containing 11 proteins (KCTD11) are co-deleted with TP53.96 Analysis of tumors at this transition from TP53Mut/wt to TP53Mut/− would be highly informative in this regard.Antimorphic or DN mutations, as subsequently described by Herskowitz,ypeptide . DN acti98 The boundaries between hypermorph and neomorph mutations are not clear cut and alterations in a single gene can lead to variants that can have either one or both characteristics.The ‘hypermorph' label is the most explicit term to describe many protein variants expressed from mutated oncogenes, as most of them are hyperactive proteins. For amplified genes such as N-myc, MDM2 or CCND1 (the gene encoding cyclin D1), or genes upregulated via chromosomal translocations such as c-myc, this definition appears to be obvious at first glance, as it refers to the abnormal accumulation of wild-type proteins . Neverth99 As discussed in the previous section, several hotspot p53 mutants are obviously neomorphic with a marked diversity in the activity gained by each variant.Although chromosomal translocations leading to fusion genes and the synthesis of chimeric proteins with novel functional specificity are the most obvious and easy-to-demonstrate neomorph mutations, it is more difficult to demonstrate missense mutations acting as neomorph mutations. Nevertheless, neomorphic mutations have recently been demonstrated in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), which are mutated in several cancer types such as glioma and AML . In bothIn vitro, mouse in vivo and clinical studies all point toward the importance of selection for oncogenic p53 mutants in human tumors. Although classification of cancer genes into oncogenes or tumor suppressor genes can be very helpful, it is becoming increasingly obvious that the frontier between the two classes is not as clear as previously thought, and that some genes may straddle these two categories. Furthermore, tissue type, genetic background and many other factors also influence the phenotype induced by a specific mutation. Nonetheless, we can predict that, except for rare cases, most cancer gene alterations will give rise to products with pleiotropic activities. Using the classification based on Muller's proposition more than 80 years ago, it is possible to establish a comprehensive and meaningful categorization of the various cancer genes that illustrates how TP53 does not fit the classical definition of a tumor suppressor gene. Beyond the rhetorical aspect of this statement, there is a genuine need to avoid an overly simplistic, binary, ‘wild-type versus inactivated' classification; defining p53 mutants as oncogenes with heterogeneous GOFs that affect multiple pathways must be considered. Designing drugs to target specific p53 mutants seems an attractive strategy, as several of these mutants are among the most frequent protein variants found in several cancer types and associated with the death of >120,000 patients worldwide. Indeed, small molecules capable of targeting hotspot mutants p.R175H or p.Y220C have been identified. Further drug discovery efforts should focus on other frequent p53 mutants, such as p.R273H, p.R248W and p.R249S. With drugs targeting a wide range of common p53 mutants in our future arsenal, our chances of efficiently fighting cancer may be greatly improved.100


Although there are some studies about the mechanism and role of p53 aggregation and amyloidosis in tumors, there still exist some controversies. In this paper, we review the mechanism of p53 amyloid fibril structure and discuss the characteristics and effects of p53 amyloid aggregation, as well as the pathogenic mechanism leading to the occurrence of aggregation in tumors. Finally, we summarize the various inhibitors targeting p53 aggregation and prion-like behavior. In conclusion, a comprehensive understanding of p53 aggregation can expand our understanding of the causes leading its loss of physiological function and that targeting p53 aggregation might be a promising therapeutic strategy for tumor therapy.Similar to neurodegenerative diseases, the concept that tumors are prion like diseases has been proposed in recent years. p53, the most well-known tumor suppressor, has been extensively studied for its expression, mutation, and function in various tumors. Currently, an interesting phenomenon of p53 prion-like aggregation has been found in several tumors, and studies have found that its pathological aggregation may lead to functional alterations and ultimately affect tumor progression. It has been demonstrated that the mechanism of p53 aggregation involves its mutation, domains, isoform, etc. In addition to p53 itself, some other factors, including Zn Prion and prion like diseases, which result from the misfolding and aggregation of prion proteins, have been demonstrated in transmissible spongiform encephalopathies and various neurodegenerative diseases. The deposited key proteins can affect the function of the central nervous system or peripheral organs. Pathological aggregation of these key proteins can spread directly within or between cells, even between the same or different species of animals, leading to a gain of toxic function and ultimately to cell death –5. In reTo date, p53 aggregation has been found in various tumors, and wild-type p53 was found to be aggregated in neuroblastoma , 17, brep53 oligomerization occurs mainly through its oligomerization domain (OD) under physiological conditions. p53 dimerization is a normal event, and tetramer is considered the major active transcriptional unit. In nontetrameric forms of p53 (monomers and possibly dimers), p53 exists in the cytoplasm. The tetramerized p53 localize in nuclear for transcriptional activation . When DNVirtually, most proteins can aggregate under extreme chemical conditions, but only a small fraction of proteins aggregate in vivo under physiological conditions . The amyp53 amyloid aggregation affects the normal function of p53 in several ways, and various independent studies have shown that the formation of mutant p53 aggregates is related to loss of function (LOF), gain of function (GOF), and dominant negative effect (DN) , 39, 40 LOF: p53 amyloid aggregates are present in a variety of human and animal tumor tissues . The losCDK6, CDK4, CDC45, PCNA, E2F2, RAD51, TIMELESS and upregulate of genes including ERBB2, MAP2K1, EGF. As well as the EMT-associated genes, which contribute to tumor invasion and metastasis can be dysregulated by p53 amyloid formation [GOF: alterations in p53 mutant function may be associated with the GOF of "emerging" target genes. For example, the first reported mutant, R175H, activated the promoter of multidrug resistance gene 1 (MDR1), which is not targeted by wild-type p53 . Amyloidormation . Thus, pormation .DN effect: p53 acts as a tetramer in cells, and it is acknowledged that DN is caused by the combination of inactive mutant and wild-type p53 in a mixed tetramer, resulting in a reduced concentration of functional p53 in the cell. p53 aggregation provides an alternative hypothesis that the DN effect of conformationally unstable mutants is induced by mutation-induced coaggregation. When Milner et al. studied R248Q mutants, they found that the mixture of amyloid oligomers and protofibrils of R248Q, like seeds, accelerates the aggregation of wild-type p53, and the aggregation isolates not only functional p53 but also other tumor suppressors and p53 homologs . The seeActually, although LOF, GOF, and DN were tend to be studied separately, they are related to each other. In addition to losing the tumor-suppressing function of wild-type p53 (LOF), mutant p53 is also found to function in a tumor-promoting manner (GOF) through DN regulation of remaining wild-type p53 or independently of wild-type p53 , 40, 47.Many mechanisms have been reported for p53 amyloid aggregation. In the following, we focus on the mechanism of p53 amyloid aggregation in terms of p53 high frequency mutations, structural domains, and isoforms  as the core region of p53 are taken for hotspot mutations . p53 agg2+ of p53 leads to exposure of this segment, which can result in p53 self-aggregation. The process is an ordered process by which the aggregation-nucleating segment self-assembles into a nonnative state of β-structured molecular assemblies [Many studies have focused on the aggregation tendency of p53 domains. The three functional domains of p53, transactivation domain (TAD), DBD, and OD, have been demonstrated to form amyloid aggregates in vitro, and the highest aggregation propensity remains in the DBD region, which has the most hotspot mutations , 46, 58.However, by analyzing aggregates by restriction protein hydrolases and the effect of mutations on kinetics and agglomeration products, Wang et al. suggested that there is no unique aggregation sequence for p53 in vitro but rather multiple sites. The occurrence of p53 aggregation is not the contribution of an individual site but a collaborative network . Stindt p53 dysfunction may also be caused by abnormal expression of isoforms. The p53 gene consists of 11 exons . ThroughAs mentioned before, a partial peptide of the p53C structural domain can induce p53 aggregation in vitro, which is similar to the N-terminal truncated p53 isoforms , and isoforms can form a complex with full-length p53, leading to its inactivation , 63. AltThe kinetics of amyloid aggregation occur slowly with an extended lag period in vitro but accelerate dramatically once seeds are formed, and the thermodynamics are altered. In vivo, studies are much more complicated due to proteases, chaperone proteins, and some fibrillar stabilizing factors, such as extracellular matrix components and other proteins. They will incorporate into amyloid aggregates. Some factors that may contribute or accelerate the production of amyloid aggregates are as follows: the overproduction of wild-type amyloid , the breMutations in key proteins: Conformational changes in key proteins lead to their pathological aggregation, ultimately resulting in deposits and disease, which is the unified pathogenesis of conformational diseases . MutatioChaperone abnormality: Protein folding is a prerequisite for normal function, while misfolding affects biological functions or even generates harmful oligomers. It is closely related to the binding of molecular chaperones and proteases in the intracellular translation process . On the Hsp, well-known chaperones, are frequently overexpressed in various tumors . Protein2+ concentration, pH, temperature, and pressure, may destabilize the side chain interactions, the original folding structure may be opened, and a new structure or polymer may be formed under other conditions [Environment and other factors: polypeptide chains seem to have a universal ability to form amyloid fibrils, but different amino acid sequences have different propensities to form amyloid fibrils . The standitions –95. It inditions . p53 aggnditions , and thenditions .) [The effect of RNA: RNA molecules could modulate of p53 aggregation and seeding was reported recently . It is p) .The concAs an important suppressor gene, p53 inactivation plays an important role in tumors, and scientists have investigated various approaches targeting p53 to recover the function of p53. For example, interfering with p53-MDM2 binding through small molecules or peptides , 89, 101Peptide inhibitor: The most notable therapy for targeting mutant p53 aggregation is ReACp53, which was developed by Wang et al. in 2016. ReACp53 is a sequence-specific peptide inhibitor that inhibits p53 mutant aggregation and tumor growth in a peptide-based approach. ReACp53 acts in the aggregation phase of p53 dynamic equilibrium to rescue the function of p53, and it targets the 252–258 region of p53. The hydrophobic isoleucine in this aggregation nucleating segment is replaced by an arginine in ReACp53, which inhibits the tendency of aggregation in this region by shielding the segment and converting the folding to a functional, wild-type-like state . The inh2+ ion that is crucial for proper protein folding and function. Zinc-free p53 exhibits an accelerated aggregation of proteins [I is a novel designed bifunctional ligand. On the one hand, iodine in LI contributes to the interaction with the hydrophobic segment; on the other hand, LI, which possesses metal chaperone activity, can restore the binding of zinc in mutant p53. It was found that LI not only restores the function of p53 by regulating the aggregation of mutant p53 and interacting with the zinc-binding fragment but also restores zinc binding using the metal chaperone approach, which greatly increases the content of zinc in cells. It increases cytotoxicity in tumor cells and ultimately restores p53 function. Combined with oxaliplatin, LI increases the effectiveness of platinum-based therapy [Bifunctional small molecule: The DBD region of p53 contains a Znproteins , 113. Mu therapy .Protein mimics: Oligopeptide-based α-helical mimics have been reported to be effective in modulating the self-assembly of the key proteins, amyloid β-peptide in Alzheimer's disease , 115 andSince p53 aggregation proceeds through multiple sites, blocking one site may not be an effective strategy to stop its aggregation. Combining physical, chemical, and computational approaches to obtain information about the initial steps of protein aggregation revealed the existence of a unique precursor state molten globular phase of p53 . The proIn addition to pathogenicity, aggregation has been reported to be functional in bacteria, fungi, and higher eukaryotes. The prevailing view of protein aggregation in the field of neurodegenerative diseases is abnormal and harmful to cells. In 2018, a study reported that aggregates of the TDP-43 protein, which occurred in most cells of neurodegenerative diseases and are considered harmful, are beneficial to healthy muscle. We may change our mind that amyloid aggregation may have beneficial effects rather than simply being associated with disease .To date, there is still much controversy regarding whether tumors can be classified as prion diseases, and many questions need to be addressed with experimental evidences. In addition to p53, scientists have also searched for the presence of other related proteins that misfold or aggregate in tumors. PTEN, which has properties similar to p53, such as a high propensity of mutation, intrinsically disordered regions (IDRs), and oligomerization ability, has been reported to have aggregation behavior recently . ComputaOver the years, scientists have comprehensively investigated p53 in many aspects, and new p53 aggregation and prion-like characteristics in tumors have been proposed in recent years. The aggregated mutant p53 in cells, the interaction with its targets, the signaling with other cells, and the effects on tumor progression represent an emerging field and new therapeutic possibilities. Elucidation of the cellular biology and structural features of p53 amyloid aggregates will be an important topic in tumor biology and will provide new perspectives and directions for targeting p53 for tumor treatments.