HER2 (p=.003)aberrations were more common in the p53 mutation group. No survival difference was observed between patients with p53 mutations and those with wild-type p53. In patients with wild-type p53 and other aberrations, patients treated with matched-therapy against the additional aberrations had longer survival compared to those treated with non-matched-therapy or those who received no therapy . Results were confirmed in a multivariate analysis (p= .0002). In the p53 mutation group with additional aberrations, those who received matched-therapy against the additional aberrations had survival similar to those treated with non-matched-therapy or those who received no therapy (p=.15). In conclusion, our results demonstrated resistance to matched-targeted therapy to the other aberrations in patients with p53 mutations and emphasize the need to overcome this resistance.P53 mutations are associated with invasive tumors in mouse models. We assessed the p53mutations and survival in patients with advanced cancer treated in the Phase I Program. Of 691 tested patients, 273 (39.5%) had p53 mutations. Patients with p53 mutations were older (p<.0001) and had higher numbers of liver metastases (p=.005). P53 mutations were associated with higher numbers of other aberrations; PTEN (p=.0005) and  The tumor suppressor protein p53 is activated in response to a variety of stress signals to ensure genome stability in cells. The role of p53 in guarding the cell reflects its ability to act as a potent transcriptional activator, regulating the expression of genes that inhibit cell cycle progression or induce apoptosis or senescence. By eliciting cell growth inhibition, p53 functions to prevent cancer development . In addiin vivo studies. Mice that carry mutant p53 develop different types of tumors with more invasive phenotypes than the p53-null mice in which the p53 protein is not expressed [Loss of p53 function occurs frequently in human cancers and results from mutations in the p53 gene or defects in the pathway that activates p53 . More thxpressed , 8. In axpressed , 9, 10. xpressed . In addixpressed , 12.Several studies have investigated the importance of p53 mutation status in predicting clinical outcome in various types of cancer. Some of the p53 mutations have been correlated with shorter survival or a poorer response to treatment in several cancers. In addiWe have previously reported the clinical characteristics and response to standard systemic therapy of 145 patients with documented p53 mutational status (n=66 mutated vs. n=79 wild-type tumors) who were referred to our department for participation in Phase I clinical trials. In the From May 2010 to April 2013, 691 patients were found to have tumor tissue tested for p53 mutation status. Of these, 273 (39.5%) had p53 mutations and 418 (60.5%) had wild-type p53. The patients' baseline characteristics by p53 mutation status are shown in Table No difference between the two groups was observed in sex, race, performance status, number of metastatic sites, number of prior therapies, serum albumin and lactate dehydrogenase levels, and Royal Marsden Hospital score. The most common tumor types seen were colorectal, head and neck, and breast cancers, which reflected the referral pattern of patients in the Phase I Clinic. Colorectal cancer was the most common malignancy in both groups .Of 273 patients with p53 mutations, 17 (6%) had more than one p53 mutation. The distribution of p53 mutations by exons, codons, and tumor types is summarized in Table When all screened patients were considered, p53 mutations in the hot-spot codons  were significantly more common in colorectal cancer  than in the other tumor types . When the analysis was limited to patients with p53 mutations, 24 (49%) patients with colorectal cancer had hot-spot mutations compared to 60 (27%) patients with the remaining tumor types (p=.0002). Mutations in the DNA-binding domain excluding the hot-spot codons were more commonly seen in ovarian and lung cancer .BRAF mutations in the patients with wild-type p53 compared with those with mutated p53 .We examined aberrations other than p53 in patients with p53 mutations and wild-type p53. Additional aberrations were found more often in the group with p53 mutations than in the wild-type p53 group (p=.002). The distribution of the number of other aberrations in the two groups is summarized in Table KRAS (16%), APC (12%), and PI3K (10%). The most common aberrations seen in the wild-type p53 group were KRAS (16%), PI3K (14%), and PTEN (9%). Overall, aberrations involved in the RAS/RAF/MEK pathway were more frequent in the p53 mutant group than in the wild-type p53 group . In contrast, no difference was observed between the two groups in aberrations involving the PI3K/AKT/mTOR pathway .The distribution of the common additional aberrations in the groups of patients with mutant and wild-type p53 is summarized in Table Of the 273 patients with p53 mutations, 195 (71%) were treated on various phase I clinical trials; among them, 20 (7%) patients received local therapy, including various hepatic arterial infusion and intra-peritoneal therapy regimens. The remaining 78 (29%) patients did not receive any treatment for various reasons. Of 418 patients with wild-type p53, 312 (75%) were treated on various phase I clinical trials; among them, 26 (6%) patients received local therapy. The remaining 106 (25%) patients did not receive any therapy. The reasons for which patients did not receive treatment were ineligibility or unavailability of a clinical trial , insurance issues , and personal reasons .Among patients with additional aberrations in the p53 mutation group, 37 (26%) patients received targeted therapy against the additional aberrations, 62 (43%) patients received non-matched therapy, and 44 (31%) patients received no therapy. In the wild-type p53 group, 78 (30%) patients received targeted therapy against the additional aberrations, 117 (45%) patients received non-matched therapy, and 63 (25%) patients received no therapy.There was no difference in overall survival between patients with p53 mutations and those with wild-type p53 . The overall survival of treated and untreated patients with p53 mutations is shown in Figure The overall survival of patients with p53 mutations by exon is shown in We performed survival analyses of patients with p53 mutations and additional aberrations. The median survival durations were 13.6 months in patients who received matched therapy (n= 37), 13.9 months in those who received non-matched therapy (n= 62), and 7.6 months in those who received no treatment n= 44)  had longer survival than those treated with non-matched therapy (n= 117) or those who received no treatment (n= 63)  confirmed this difference between the treatment groups (p=.0002). These findings translated into longer survival for the patients with a more number of additional mutations in the wild-type p53, but not the mutant p53 group .This analysis expands on our previous report on the characteristics and survival of patients with advanced cancer and p53 mutations. In the current study, we evaluated the effects of specific p53 mutations and additional aberrations on survival in patients referred to the Phase I Clinical Trials Program. P53 mutations were associated with higher numbers of other aberrations and resistance to targeted therapy. P53 status did not affect overall survival in this patient population, but matched therapy for other aberrations was associated with longer survival in patients with wild-type p53. P53 mutations varied by tumor type and were associated with higher proportions of PTEN and HER2 aberrations than wild-type p53.Overall, 39.5% of the patients analyzed had a p53 mutation. Patients with p53 mutations were older (p<.0001) and had higher rates of hepatic metastases (p=.005) than patients with wild-type p53. P53 mutations within exon 5 were more common in colorectal cancer, while mutations within exons 6, 7, and 8 were more common in lung, pancreatic, and ovarian cancer, respectively.Our findings of older age, more frequent liver metastases, and more frequent PTEN loss in the p53-mutated group in the current, more extensive analysis, were similar to those in our prior analysis .Our results are consistent with previously known data showing that the percentage of p53 mutation varies by tumor type and ranges from 10% to 80% . Our resThe observation that patients with p53 mutations had more liver metastases may be partially explained by the role of mutant p53 in promoting cell migration and invasion , as showThe distribution of various types of p53 mutations in our study varies by tumor type. This difference may contribute to the variable prognostic value of p53 mutations in various tumor types with p53 mutations .In our series, tumors with p53 mutations had more additional aberrations than tumors with wild-type p53 (p=.002) . In both groups, the overall survival duration was longer in patients who received treatment than in those who did not. However, among patients with wild-type p53 and other aberrations, survival was superior in those who received matched therapy against the additional aberrations compared to non-matched therapy. We have previously published evidence that identifying specific molecular abnormalities and choosing therapy based on these abnormalities is associated with a longer time to treatment failure in the phase I setting than that of previous systemic therapy. Furthermore, in the non-randomized setting, rates of response, time to treatment failure, and survival were higher with matched targeted therapy than with non-matched therapy .An intriguing finding in the current report is the observation that patients with wild-type p53 and other aberrations who received matched therapy against these aberrations had longer survival than those treated with non-matched therapy or those who received no treatment . This survival advantage was confirmed in multivariate analysis after adjustment for other covariates (p=.0002). In contrast, in the p53 mutation group with additional aberrations, the survival durations were similar for those who received matched therapy against the additional aberrations, those treated with non-matched therapy, and those who received no therapy .The exact role of p53 in the context of other molecular aberrations and their prognostic significance in various tumor types needs to be elucidated. Whether p53 mutations are driver mutations or contribute to the emergence of resistance to targeted agents needs to be evaluated. Resistance to therapy seen in tumors with p53 mutation requires the development of new agents or strategies. Several investigators have reported that p53-based cyclotherapy may represent a successful strategy. -30 This Our data suggest that the identification of driver mutations in patients with wild-type p53 and other aberrations and the selection of targeted therapy may contribute to improved survival. Recently published data in head and neck squamous cell carcinoma also demonstrated that PI3K/Akt/mTOR inhibition using PF-04691502 is enhanced with induction of wild-type p53 in human xenograft and murine knockout models . The autIn conclusion, our data add to the published data demonstrating that p53 mutations are associated with a poor prognosis and resistance to treatment, and they emphasize the need to develop agents or strategies to overcome this resistance. These findings further support the need to individualize cancer therapy and should be validated in carefully designed prospective trials.We reviewed all patients who underwent testing for p53 mutation status in the Department of Investigational Cancer Therapeutics at MD Anderson Cancer Center. Molecular profiling was performed on available tissue samples from consecutive patients with advanced tumors referred to the Clinical Center for Targeted Therapy. Patients were of various ages and had advanced or metastatic cancer that was refractory to standard therapy, that had relapsed after standard therapy, or for which there was no standard therapy available. All protocols required that participants have evidence of evaluable or measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) guidelines , 33 and The p53 mutation status was determined by either polymerase chain reaction–based or next-generation sequencing in a Clinical Laboratory Improvement Amendments–certified laboratory, as previously described . The nexTreatment was selected as previously published . BrieflyPatients' characteristics were analyzed using descriptive statistics. Categorical data were described using contingency tables, including counts and percentages. Continuously scaled measures were summarized by median and range. The association between two categorical variables was examined using the chi-square test. Survival and hazard functions were estimated using the Kaplan-Meier method, and survival between groups was compared using the two-sided log-rank test. Survival was analyzed according to the type of p53 mutations and the treatment. Hazard ratios with and without adjustment for potential confounding variables were estimated using Cox proportional hazard regression analysis.


Progression through the cell cycle is one of the most important decisions during the life of a cell and several kinds of stress are able to influence this choice. p57 is a cyclin-dependent kinase inhibitor belonging to the CIP/KIP family and is a well-known regulator of the cell cycle during embryogenesis and tissue differentiation. p57 loss has been reported in a variety of cancers and great effort has been spent during the past years studying the mechanisms of p57 regulation and the effects of p57 reexpression on tumor growth. Recently, growing amount of evidence points out that p57 has a specific function in cell cycle regulation upon cellular stress that is only partially shared by the other CIP/KIP inhibitors p21 and p27. Furthermore, it is nowadays emerging that p57 plays a role in the induction of apoptosis and senescence after cellular stress independently of its cell cycle related functions. This review focuses on the contribution that p57 holds in regulating cell cycle arrest, apoptosis, and senescence after cellular stress with particular attention to the response of cancer cells. Cells can encounter different kinds of stress during their life and in turn have evolved a wide range of responses. Stress-activated signalling pathways such as ATM/ATR, JNK/SAPK, and p38 pathways are activated in mammalian cells by DNA damage, starvation, heat and osmotic shock, and oxidative stress. Depending on the kind, severity and duration of insult, and on the cell type, these responses can lead to different final outcomes, spanning from cell survival to cell death. Cell cycle delay or arrest is often the first safety step triggered in a stressed cell, followed by injury repair and thus restoration of cellular proliferation, or by the induction of cellular senescence or cell death. in vitro, called for this reason replicative senescence. Genotoxic stress and more generally prolonged activation of the DNA damage response pathways results in the so-called premature senescence. Interestingly, cells usually arrest cell cycle in G1 phase during replicative senescence and in G2 phase during premature senescence. Senescent cells often display a flat, enlarged morphology and exhibit an increase in the lysosomal β-galactosidase activity that can be used as senescence biomarker . Many senescent cells also display a characteristic senescence-associated secretory phenotype (SASP)  is considered a master regulator of the cell cycle during embryogenesis and tissue differentiation , 14, butp57 belongs to the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) along with p21 and p27. The CIP/KIP family counteracts cell cycle progression inhibiting all the cyclin CDK complexes throughout the cell cycle (for a review on CKIs see ). In parDue to its role in cell cycle control, p57 is involved in the regulation of many cellular processes such as embryogenesis and tissue differentiation. In muscle differentiation p57 participates in the regulation of cell cycle exit of differentiating myoblasts , 25. Durp57 is considered a tumor suppressor gene due to its ability to inhibit proliferation and the importance of p57 in the suppression of cancer is highlighted by its mutation/inactivation in the Beckwith-Wiedemann Syndrome, a cancer predisposing syndrome . RemarkaA cytoplasmic localization for p57 has been described in non-small-cell lung carcinoma and esophageal squamous cell carcinoma , 42, sug cdkn1c, is located in the imprinted domain kcnq1/kcnq1ot1. Maternal and paternal allele of an imprinted gene display, despite the fact that they have identical sequences, different epigenetic modifications, such as DNA methylation within CpG islands, histones acetylation, and methylation. The imprinting of a cluster of genes is regulated by specific sequences acting in cis, known as imprinted control regions (ICR). In particular, cdkn1c is expressed only by the maternal allele, while the paternal allele is silent  have been reported to downregulate the expression of cdkn1c . For exaFinally, p57 protein stability is regulated by both phosphorylation and ubiquitination . In partAll the three members of the CIP/KIP family play an important role in controlling cell cycle exit. In particular, p57 is a master regulator of the cell cycle during embryogenesis and tissue differentiation , 14, butA CDK inhibition-dependent mechanism has been described for the stress-activated protein kinase (SAPK) p38 signalling, which is activated in mammalian cells by several insults, such as osmostress, oxidative stress, ionomycin, and UV –58. It h2-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway with a CDK inhibition-independent mechanism. Indeed, p57 negatively regulates the JNK/SAPK signalling cascade through direct inhibition of JNK/SAPK, independently of its well-known inhibitory function on CDKs [On the other hand, p57 participates in the c-Jun NH on CDKs . Deletio on CDKs , while t on CDKs , 62. Pre on CDKs , 64. It L or Bcl-2 was found to counteract p57 proapoptotic effect. The molecular mechanism by which p57 promotes cell death was investigated by Vlachos and colleagues [c release into cytosol and consequent caspase-9 and caspase-3 activation. Mitochondrial pathway specificity was confirmed, as p57 expression was ineffective in promoting death receptor-mediated apoptosis stimulated with agonistic anti-FAS antibodies. The mechanism by which p57 triggers the mitochondrial intrinsic pathway has been linked to the ability of p57 to stabilize the actin cytoskeleton by Kavanagh and colleagues [p21 and p27, the other members of the CIP/KIP family, have been reported to play a role in apoptosis , 66 and lleagues . p57 ovelleagues . They delleagues . Furtherβ-mediated apoptosis induced by cisplatin treatment [p57 proapoptotic effects were observed in different cell lines, including HeLa cervical cancer cells, SH-SY5Y neuroblastoma cells, SKOV3 ovarian carcinoma cells, and mouse embryonic fibroblast cells . In H129reatment . Howeverβ, lipopolysaccharide, tumor necrosis factor-α, insulin-like growth factor 1, and different forms of cellular stress such as high-density culture or serum withdrawal. They found p57 reactivation only in cell lines with unmethylated promoter but not in methylated cells, leading to different outcome ranging from no effect on cell growth to G2 arrest and apoptosis, depending on cell type and type of insult. Remarkably, exogenous overexpression of p57 in p57 promoter-methylated leukemic cell lines resulted in marked cell growth arrest and induction of apoptosis, while the overexpression of p57 in partially methylated cells only resulted in a moderate inhibition of cell growth and had no impact on apoptosis, suggesting that the epigenetic status of p57 promoter can influence the cell response to stress stimuli.Kuang and colleagues performed an interesting study on different leukemia cell lines that differ for p57 promoter methylation status . They anIt is worthy of notice that the proapoptotic effects of p57 described so far have been mainly reported in cancer cells after p57 overexpression or reinduction, while antiapoptotic activity of p57 has been observed in its physiological regulation of JNK pathway (see previous section) and during embryogenesis, suggesting that cellular context drives a major contribution to the final outcome. For example, antiapoptotic activity of p57 was reported in response to green tea polyphenols administration. Catechins are known to induce cell death in many types of tumor cells, but normal human epithelial cells were found to survive in the presence of polyphenols because of their ability to induce p57 expression. p57 was able to prevent green tea polyphenols-induced Apaf-1 expression thus avoiding apoptosis . Finally Hes1. p57 infected cells or Notch1- and Notch3-silenced cells, which upregulates p57, arrest growth with a senescent morphology, SA-β-gal staining, and p16 expression [In two human hepatocarcinoma cell lines (HepG2 and SNU398) p57 overexpression has been found to affect proliferation and morphology without affecting the apoptotic machinery. In these cells, p57 expression is regulated by neither miR-221 nor the methylation status of the promoter but instead by the Notch target genepression .β-gal activity was reported to be partially reversible withdrawing p57 forced expression. Although senescent cells are thought to be resistant to apoptotic cell death, in one of the astrocytoma cell lines induced to express p57 (U373), a small subset of cells (15% of the population) was described to undergo apoptosis. Notably, Bax levels were unchanged. Why this occurs and why this particular cell line responds in a different manner to p57 induction is an interesting yet unanswered question.In a similar manner, Tsugu and colleagues have shown that p57 induction in p57-negative human astrocytoma cell lines  can block the proliferation and alter the morphology, with cells becoming large and flat with an expanded cytoplasm . These fA pivotal role of p57 in the premature senescence of vascular smooth muscle cells has been shown by Valcheva and colleagues in vitamin D receptor knockout mice . InteresIs the final outcome dependent on p57 levels? in vitro studies and overexpression of any gene can lead to experimental artefacts, which is the physiological relevance of p57 induction in vivo?As many data come fromWhich is the grade of overlapping between the three members of the CIP/KIP family?Bearing in mind that stopping abnormal proliferation is a key goal of our scientific community, is the reinduction of p57 a promising approach for cancer therapy?Do cancer cells respond in a different way from normal cells to p57 overexpression?Initially identified as a cyclin-dependent kinase inhibitor, p57 has since been shown to have different cellular roles aside from cell cycle inhibition, such as cell migration and regulation of cell differentiation. Nowadays it is emerging that p57 plays a key role also in coordinating the cellular response to stress, being able to drive to both apoptosis and cellular senescence. Different mechanisms of action, both CDK inhibition-dependent and CDK inhibition-independent, have been disclosed and, as highlighted in this review, p57 is now implicated in the crosstalk between several different pathways, among which MAPK signalling, DNA damage response, mitochondrial apoptotic pathway, and cytoskeleton organization. The findings that p57 can induce cell cycle arrest, apoptosis, or cellular senescence depending on cell types and cellular context arise several questions:p57 is now emerging as a new master regulator of cell fate and the mechanisms through which p57 participates in the cellular response to stress have been just started to be dissected.


The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.The functional importance of  Numeroueatments . In addi did not . Cells esiveness , 21. Mutisplatin . In Li–Futations . These np53 knockin alleles in mice provided direct in vivo evidence for the GOF activities of mutant p53. Knockin mouse models that express mutant p53R172H and p53R270H proteins, which mimic hot spot mutations that correspond to amino acids 175 and 273 in human cancers, respectively, develop tumors that exhibit a GOF phenotype in vivo, with high metastatic capacity compared to tumors in mice inheriting a p53-null allele  incidence of metastasis in mice expressing a single, mutant p53 allele  and inhibited the function of p63, properties that were essential for the invasive nature of these cells . Mutant p53-dependent sequestration of p73 from an NF-Y complex permits this transcriptional complex to function at the platelet-derived growth factor β promoter, resulting in expression of PDGFRβ and a prometastatic phenotype  breast cancer cells, Freed-Pastor et al.  p53, bind to and upregulate chromatin regulatory genes, including the methyltransferase MLL1, MLL2, and acetyltransferase MOZ, resulting in genome-wide increases of histone methylation and acetylation. Furthermore, upregulation of MLL1, MLL2, and MOZ was found in human tumors with p53 mutants, but not in WT p53 or p53-null tumors (p53. The emerging themes by which mutant p53 exhibits its GOF are (1) through formation of mutant p53 complexes with other proteins that modify their activities  and (2) by interaction of mutant p53 with other transcription factors  that bring a potent transcriptional activation domain to promoters not normally regulated WT p53 -on-chip experiments and expression arrays using SKBR3 breast cancer cells with the ic genes . Importar et al.  identifi changes . In this culture . Lastly,tant p53 , 44. Morl tumors . In summ3 Figure , 47.p53 proteins exhibit intrinsic differences. Some are classified as structural mutants  as the mutation alters the structure of the DNA-binding domain while others are classified as DNA-binding mutants  because they alter an arginine that directly interacts with DNA. Other mutants show partial defects. For example, the p53R172P mutation, albeit rare, is able to activate the cell cycle arrest but not apoptotic programs of p53  deposition, wound healing, and chronic inflammation, which were validated by staining with various markers. Lujambio and colleagues selectively deleted p53 in hepatic stellate cells, resulting in modifications to the tumor microenvironment (TME) and enhanced malignant transformation of epithelial cells   , 60–63. ID hosts . Hill etID hosts  further p53 proteins exhibit GOF activities, a growing body of evidence suggests that tumor cells may be addicted to mutant p53 expression. Experiments using siRNA knockdown of mutant p53 in cancer cell lines showed a higher apoptotic response to drug treatment in cells with knockdown of mutant p53  in 3D culture leads to phenotypic reversion to more normal, differentiated structures with hollow lumens  or whey acidic protein (WAP) promoters which are hormonally regulated and therefore do not simulate expression of mutant p53 from the endogenous locus   replete with p53-heterozygous stromal elements. CAFs, macrophages, T cells, neutrophils, endothelial cells, and so on remain heterozygous for p53 following conception (due to the presence of the STOP cassette) with undefined effects on tumor biology. Stroma is a requisite component of tumor initiation and growth, and as mentioned earlier, prostate tumor epithelium selects for p53-null stromal fibroblasts in p53± mice, yielding a highly proliferative stroma that contributes to tumor progression. Given the powerful roles of p53 in cellular plasticity and embryonic stem cell differentiation, tumors that develop from and under conditions of p53 heterozygosity may differ in unappreciated ways from human tumors that initiate from WT p53 cells. A model that more closely mimics the events in sporadic tumorigenesis is sorely needed.Knock-in mice with germline mutations in tivities , 25. Yetus locus , 71. Curp53 carry the potential to inform us of essential mechanisms of cancer initiation and metastasis translatable to therapeutics in humans. However, many genetic mouse models used to study mutant p53 in vivo incorporate germline mutant p53 alleles that may alter normal and cancer biology in ways that compromise its relevance to human cancer. Innovation in genetic mouse models of mutant p53 is mandatory to more closely model human biology and to serve as translational biologic platforms to better evaluate and develop novel therapeutic agents in human cancers. Moreover, given the importance of the TME in cancer development and metastasis, mouse models that preserve the complex regulatory and tumor–stromal interactions are mandatory to the development of effective, translational biologic platforms to target the TME toward therapeutic ends.Mouse models of mutant MK, YZ, and GL wrote and edited the manuscript. MK and YZ generated the figure.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.


About 15 years ago it was proposed that generation of the truncated protein p25 contributes to toxicity in Alzheimer’s disease (AD). p25 is a calcium-dependent degradation product of p35, the principal activator of cyclin-dependent kinase 5 (Cdk5). The biochemical properties of p25 suggested that its generation would cause Cdk5 overactivation and tau hyperphosphorylation, a prerequisite for neurofibrillary tangle (NFT) formation. Whilst this model was appealing as it explained NFT formation, many laboratories could not confirm the finding of increased p25 generation in brain from AD patients. On the contrary, it emerged that p25 levels are reduced in AD. This reduction occurs primarily in the early stages of the disease. Further, p25 generation in the mouse hippocampus is associated with normal memory formation and p25 overexpression enhances synaptogenesis. Therefore, it transpires that p25 generation is a molecular memory mechanism that is impaired in early AD. I discuss the prospect that investigation of p25-regulated proteins will shed light into mechanisms underlying synaptic degeneration associated with memory decline in AD. Figure 1) was as follows: Abnormal APP processing generates amyloid peptides that oligomerize thereby enhancing calcium signaling (In the late Nineties Li-Huei Tsai’s lab published a paper proposing that the truncated cyclin-dependent kinase 5 (Cdk5) activator p25 is specifically formed in sporadic Alzheimer’s disease AD; . This fiignaling . This woignaling . p25 forignaling . Additiodownregulation of p25 expression in AD forebrain. This decreased p25 expression was not simply due to neuronal loss, as it was adjusted to the expression of a neuronal marker. However, the downregulation of p25 expression was not discussed by the authors, possibly because p25 expression was considered toxic in line with Tsai’s lab. Instead, the key point of discussion was that p25 upregulation was not reproducible in postmortem AD tissue. It was realized that postmortem delay is a variable to consider, since p25 can be formed from degradation of p35 before the tissue is frozen (p = 0.06)   and in t = 0.06) . Recentl = 0.06) . In our  = 0.06) , but we  = 0.06) . Taken t = 0.06) .Motivated by the How can p25 expression be reduced in AD? A reduction in p25 expression indicates that p25 formation occurs under normal physiological conditions, and that such physiological p25 generation is impaired in AD. In agreement with this, spatial memory formation is associated with p25 generation in the mouse hippocampus . ImportaThe importance of p25 generation for spatial memory has been tested in various mutant mouse lines. First, mutant mice expressing low levels of p25 under control of the αCaMKII promoter which is active in postnatal forebrain, show improved spatial memory formation . Second,Overexpression of physiological levels of p25, or transient expression of high levels of p25 enhance spatial memory formation . In thesIn AD synapse loss, which precedes neuronal loss, correlates best with impaired memory formation . Thus, sCdk5 and glycogen synthase 3 (GSK-3) are thought to be the main tau kinases . In AD rFigure 1). Impaired p25 generation in AD is expected to contribute to synaptic degeneration in the disease. However, more studies are needed to establish the precise synaptic function of p25. For example, p25 generation could be a signal from synapse to nucleus to stimulate gene expression needed for long-lasting synaptic plasticity. Recent evidence also shows that the expression of p25-regulated synaptic proteins is downregulated in early AD hippocampus. Analysis of these synaptic downregulations promises to give insight into the mechanisms of early synaptic degeneration in AD. So far, it has emerged that p25-regulated proteins are downregulated at the pre- and post-synaptic side, suggesting that degeneration occurs equally at both sides of the synapse. This is consistent with the finding that oligomeric amyloid peptide binds to pre- and post-synapses and has the potential to induce dysfunction on both sides of the synaptic cleft (In recent years it has emerged that p25 is not an exclusively toxic molecule. Spatial memory formation leads to p25 generation in the hippocampus, where p25 can stimulate synthesis of synaptic proteins followed by synaptogenesis and synaptic strengthening. In the early stages of AD hippocampal p25 expression is reduced. Taken together, this suggests that p25 generation is a memory mechanism that is affected in early AD (ic cleft . It is hThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


JASPAR (profile score threshold 80%) and TFSEARCH datasets were used to search transcriptional start sites for p53/p63 response elements. Remarkably, these data revealed 63 microRNAs that targeted p63. Furthermore, there were 39 microRNAs targeting p63 that were predicted to be regulated by p63. These analyses suggest a crosstalk between p63 and microRNAs. Here, we discuss the crosstalk between p63 and the microRNA network, and the role of their interactions in cancer.The transcription factor p63 is a member of the p53 gene family that plays a complex role in cancer due to its involvement in epithelial differentiation, cell cycle arrest and apoptosis. MicroRNAs are a class of small, non-coding RNAs with an important regulatory role in various cellular processes, as well as in the development and progression of cancer. A number of microRNAs have been shown to function as transcriptional targets of p63. Conversely, microRNAs also can modulate the expression and activity of p63. However, the p63–microRNA regulatory circuit has not been addressed in depth so far. Here, computational genomic analysis was performed using miRtarBase, Targetscan,  The regulation of gene expression is implicated in multiple molecular mechanisms, which include transcriptional, translational, and post-translational regulatory mechanisms , 2. In aP63 is a member of the p53 gene family of transcription factors . Like otAlthough p63 has been shown to closely associate with cancer development, the underlying molecular mechanisms have not been fully elucidated. In fact, many studies indicate that transcriptional regulation of p63 is one of the most important mechanisms in various tumor processes by triggering the expression of oncogenes , 23. TApmiRNAs are 18- to 24-nucleotide non-coding RNA molecules . It regumiRNAs are canonically produced through a multistep process and transcription factors are recognized as key regulatory points for miRNA production . AbnormaThe human p63 gene belongs to the p53 family and plays a crucial role in maintaining genomic stability and suppressing tumor formation . AlthougThe p63 gene contains two promoters and produces two diametrically opposite groups of isoforms: those containing the transactivation (TA) domain (TAp63), and those lacking it (ΔNp63) . ΔNp63 iTAp63 is transcribed from the first transcriptional start site within the p63 gene and contains the transactivation domain and three different C-termini termed α, β and γ . The higThe role of p63 in tumorigenesis is very complex. Thus, more work is needed to explain how the differential expression of the p63 isoforms could influence this process. Considering the fact that all p63 isoforms contain a DNA binding domain and are able to bind to thousands of gene promoters, studying their interaction with regulatory genes may not only increase our understanding of the role of p63 in tumorigenesis, but could also open the door to the development of innovative diagnostic procedures and targeted therapies.miRNAs are small, non coding RNAs with a length of 18 to 24 nucleotides . They plmiRNAs are encoded throughout the human genome. Most miRNAs are found within intragenic regions as well as protein-coding or non-coding transcriptional units . The proAs discussed previously, miRNA biogenesis is a complex multistep process, and each step of this process is tightly regulated. Mutation or aberrant expression of any component of the miRNA biogenesis machinery could contribute to a dysregulation in miRNA expression in tumors. The majority of miRNAs are transcribed by RNA polymerase II and III, and transcription factors significantly contribute to the activation of their expression by directly binding to the promoters of miRNAs. Recent evidence indicates that p63-mediated activation of miRNA transcription is an important event in the regulation of miRNA levels , 95.Dysregulation of miRNA expression is common in human cancers. However, the mechanisms underlying miRNA dysregulation are not clear. As described above, transcriptional regulation is one of the most important steps in the synthesis of miRNAs. Transcriptional activation and repression of specific microRNAs by ΔNp63α was shown to attenuate the expression of several proteins involved in cell death and survival and to regulate tumor cell resistance to cisplatin . In humaAnother important transcriptional program that has been identified involves post-transcriptional control of miRNAs by p63. The cropping step regulated by the Drosha/DGCR8 heterodimer is an important event in regulation of miRNA levels. There has been no direct study to prove that p63 can directly regulate the expression of Drosha. However, it has been reported that PY domain-containing proteins interact with WW domain-containing proteins. Interestingly, p63 contains a PY domain , while DFurthermore, p63 can control the expression of miRNAs by regulating the expression of miRNA transcription factors, as well as several key regulatory factors that allow for the correct maturation of primary miRNA. As an example of the former, the gene of the transcription factor early growth response 2 (EGR2) contains a p63 RE and is therefore a direct target of p63 . InteresAs discussed above, p63 has been defined as a regulator of miRNAs. Conversely, p63 expression can also be controlled by specific miRNAs in certain tissues. Recent studies identified p63 as one of the conserved targets of miR-203 –114, andAdditionally, Papagiannakopoulos T et al. showed for the first time that miR-21 targets the tumor-suppressive protein TAp63, and confirmed that miR-21 is necessary for suppression of apoptosis in glioblastoma cells . Wang T Given these findings, p63 isoforms regulated by miRNAs can be divided into two classes: ΔNp63-isoforms, which enhance cell proliferation and repress apoptosis and are repressed by miR-92 and miR-203, and Tap63-isoforms, which induce cell cycle arrest and apoptosis and are repressed by miR-21, miR-30 and miR-302  have been validated , 130. A It is now apparent that many tumors are dependent upon a dysregulation of p63 and miRNAs, providing a rationale for therapeutic intervention of p63 and miRNAs. Therefore, the p63/miRNA network represents a novel approach for treatment and is likely to lead to new therapeutic anticancer strategies.


Lymph node metastases indicate increased tumor burden and tumor cell heterogeneity and affect both the treatment strategies and the prognosis. In this study, expressions of NF-κB, MMP-1, p53, and Ki-67 were between the primary tumors and lymph node metastases in 110 Dukes' stage C, CRC cases by immunohistochemical methods, related to patients' clinical outcomes. NF-κB, p53, and Ki-67 expressions were significantly higher in the metastatic lymph nodes compared to the primary tumor tissues . In the metastatic lymph nodes NF-κB expression was correlated with both p53  and Ki-67  expressions. The univariant and multivariant analyses showed that only “pT stage” preserved an independent prognostic significance for recurrence-free survival rates and 5-year overall survival rates (P < 0.001 for both). Metastatic cells can acquire different biological characteristics compared to their primaries. Elucidation of properties acquired by metastatic cells is important in order to better determine prognosis, reverse drug resistance, and discover new treatment alternatives.Colorectal cancer (CRC) is the third most frequent malignancy. Many factors such as NF- Colorectal cancer (CRC) is one of the most common malignancies that has high morbidity and mortality throughout the World, especially in developed countries , 2. ExisThe variations in the biological characteristics of tumor cells in the metastatic tissues have an influence on their ability to survive . A varieκB signaling is of importance to cell proliferation, apoptosis, and creation of the inflammatory response and has been found to be implicated in the development of many cancers [κB affects CRC development and especially carcinogenesis after chronic inflammations. Several studies have revealed that NF-κB can be used as a marker of resistance to adjuvant chemotherapy in CRC [NF- cancers . There hy in CRC –14.κB and MMP. NF-κB activation has been reported to be involved in the regulation of some MMP types [There is also evidence that matrix metalloproteinases (MMPs) might be a modulator of tumor progression and spread, which affect the prognosis of the disease –17. In aMP types , 24.It has been shown that mutations of p53, as a well-defined tumor suppressor gene, contribute to the development of many cancer types and that p53 is a poor prognostic factor in many cancer types –30. It wκB, MMP, p53, and Ki-67 production and the clinical behavior of metastatic tumor cells.Ki-67 is a proliferation marker, observed in all phases of cell cycle except G0. High Ki-67 expression is a poor prognostic factor in many tumors and there are correlations between Ki-67 expression and expression of other poor prognostic factors. In a previous study, Ki-67 expression was found to be higher in metastatic lymph node tissues compared to primary in breast cancer . In CRC,κB, MMP-1, p53, and Ki-67 expressions in the primary tumor tissues and metastatic lymph nodes were compared in Dukes' stage C, CRC cases.A series of unique biological features may affect the ability of cancer cells to create metastasis. It is important to reveal these biological characteristics of metastatic cells in CRC in terms of development of new therapeutic strategies. However, to our knowledge, studies are needed on the variations of expression patterns of these markers in the metastatic lymph nodes in CRC cases. Therefore, in this study, NF-A total of 457 CRC patients were operated on in Adnan Menderes University Hospital, Aydin, Turkey, between August 1998 and December 2010. Of these, 152 with stage Dukes' C were evaluated in this study; however, 42 were excluded as either their paraffin blocks were not available for pathologic review and/or their clinical follow-up data were incomplete. None of the patients had received chemotherapy and/or radiotherapy in the preoperative period. There was no history of previous cancer, familial polyposis coli, or preexisting inflammatory bowel diseases. Preoperative evaluations, based on clinical examination, complete blood count, blood biochemistry, chest X-ray and/or computed tomography (CT), abdominal ultrasonography and/or CT, and bone scintigraphy did not show distant metastasis in any of the patients. Ethical approval was obtained from the ethical committee of the university (ADUBAPTPF-12.0141). Tissue samples from both the primary tumors and the metastatic lymph nodes were obtained in all 110 patients. The selected samples did not include the areas of necrosis, hemorrhage, inflammation, or technical deformation. The samples from the primary tumors were representative of the histological grade of each tumor and the samples from the lymph nodes (LN) were obtained from the largest area of metastases. Microscopic examinations of the haematoxylin-eosin- (HE-) stained slides obtained from the paraffin blocks were used to confirm the diagnoses.μm in thickness obtained from selected paraffin embedded blocks were put on positively charged slides. Second, all the sections were deparaffinized in xylene and dehydrated through a graded series of ethanol solution. Third, antigen retrieval was performed at 96°C  for 40 minutes in a thermostatic bath (PT Link). The sections were incubated with NF-κB/p50 , MMP-1 , anti-Ki-67 monoclonal antibody MIB-1 , and anti-p53  for 60 minutes at room temperature. Positive and negative controls were added for each antibody and to each batch of staining. A streptavidin-biotin enhanced immunoperoxidase technique  in an automated system was used to show immunoreactions. The sections were incubated with DAB and counterstained lightly with haematoxylin to demonstrate binding. Finally, the sections were dehydrated and mounted onto the slides. The slights known to be positively immunostained were used as positive controls. Normal rabbit serum IgG was used to replace primary antibody as a negative control.All immunostaining was carried out at room temperature using DAKO Autostainer Universal Staining System . First, sections 4 Sections were reviewed by 3 pathologists , who were blinded to the patients' clinicopathological characteristics and follow-up information, and discrepancies (about 12% of cases) were resolved by joint discussion of the slides viewed with a multiheaded microscope. Immunostaining was scored (as described by Zhigang and Wenlv) based on the intensity of staining and the percentage of cells that stained positively . The intP < 0.05) to identify the independent factors of recurrence. Survival rates were calculated by the Kaplan-Meier method and compared by the log-rank test. Stepwise forward Cox regression model was conducted for parameters found to be significantly associated with survival by the log-rank test (P < 0.05) in order to identify the independent factors of survival. Values of P < 0.05 were considered significant in all analyses.All statistical analyses were performed with SPSS . The Spearman correlation test was used to determine correlations between the expression of biological markers in the primary tumor and the expression of these markers in the lymph nodes. The Chi-squared test or Fisher's exact probability test was used to compare the recurrence rates. Logistic regression analysis was conducted on the parameters found to be significantly associated with recurrence by the Chi-squared tests or Fisher's exact probability test . However, the rate of cells stained with MMP-1 was not statistically different between the primary tumor and the metastatic lymph nodes (P = 0.07).The rate of cells stained (moderate + strong) with NF-κB expression was significantly correlated with p53  and Ki-67 expression  in the metastatic lymph node tissues. However, NF-κB expression was not correlated with p53  and Ki-67  expression in the primary tumor tissues. MMP-1 expression was not correlated with any markers in the primary and the metastatic tumor tissues. Likewise, there was not a significant difference between p53 and Ki-67 expressions.NF-r = 0.518, P = 0.018) was significant. Similarly, the size of the primary tumor was correlated with Ki-67 staining in the primary tumors . Furthermore, expressions of the other markers in the primary tumors and metastatic lymph nodes were not associated with any clinicopathological parameters.The correlation between immunohistochemical Ki-67 staining in the primary tumors and the number of metastatic nodes (P = 0.01) and 5-year overall survival (OS) (P = 0.01) rates. No significant correlations were observed among other clinical parameters and the immunohistochemical staining scores of the studied markers. In the univariant and multivariant analyses only “pT stage” of the tumor was a significant independent prognostic factor for both RFS and 5-year OS rates   the 5-year overall survival rates were 46.8%. Recurrence was observed in 42 patients (38.2%) and 68 (61.8%) had died during the follow-up. The recurrence rates in pT2, pT3, and pT4 tumors were 22.2%, 33.3%, and 55.2%, respectively. The 5-year overall survival rates in pT2, pT3, and pT4 tumors were 69.9%, 55.3%, and 32.8%, respectively. There were statistically significant correlations between the pT stage and both recurrence-free survival (RFS)  .Although CRC is one of the most common malignancies, carcinogenesis and many factors influencing the clinical course of this disease have not been completely described. Understanding the pathogenesis and the factors affecting the development and the progression of CRC would result in better prediction of the disease course and selection of appropriate targeted treatments for patients refractory to classical chemotherapy and radiotherapy . It is oκB has an important role in many physiological events such as apoptosis, inflammation, and cell proliferation. Its activation has a regulatory role in expression of more than 400 gene products associated with inflammation, cell survival, proliferation, invasion, and angiogenesis [κB in hematological and solid tumors, previously [κB have been shown to have a relation with the development and the progression of solid tumors. Aberrant NF-κB has been shown to be effective through increased proliferation and antiapoptotic mechanisms in colon cancer development as in many other types of cancer. NF-κB is known to be associated with CRC development by increasing inflammation [κB expression was higher in the lymph node metastases than in the primary tumors, which is consistent with the results of other previous studies showing that NF-κB plays a role in cancer development and progression [κB expressions in CRC. Increased NF-κB expression in lymph node metastases indicates a potential role of this molecule in the metastasis process of CRC. Although NF-κB is thought to play a role in early stages of CRC development, the idea that it can also be involved in later stages and metastases is supported by the findings of the present study.NF-ogenesis –12 Many eviously . The subammation . In the gression . The resκB. In one study, the rate of p53 overexpression was as high as 73% in primary CRC and 66% in distant metastases [κB. Actually, the relation between NF-κB and p53 is defined well. Increased NF-κB and p53 expressions in metastatic cells lend support for the idea that these two molecules are multifunctional coregulators. The proliferation marker Ki-67 is utilized as a prognostic indicator, and it is suggested that a biomarker-based approach can be an effective strategy to improve results of adjuvant treatments [Another finding of the present study is that p53 and Ki-67 expressions were significantly higher in the metastatic cells than in the primary tumor cells. As in many other tumors, in CRC, mutations which may occur in the p53 gene may disrupt the activity of NF-tastases . The p53tastases , 27. Howtastases . The preeatments . The resκB with MMPs suggesting that migratory genes may support NF-κB in inflammation and carcinogenesis [κB triggers many extracellular events like tumor angiogenesis, it is very likely to have a relation with MMP-1. In the current study, a positive correlation was not found between MMP-1 and NF-κB expressions, and also MMP-1 expression was decreased nonsignificantly in the metastatic tumors. This may be partly related to the complex stromal structures of metastatic lymph nodes and their microenvironment [MMP-1 is frequently found in aggressive tumors. It may not be the basic factor in the initial stage of tumor formation and invasion; however, it plays an effective role in advanced stages of tumors. MMP-1 is the leading enzyme which plays a role in destruction of collagens 1, 2, and 3, the most common stromal structures between the tissues. It has an active role in various tumors such as gastric cancer, breast cancer, squamous cell carcinomas of the head and the neck, colon carcinomas, and adenocarcinomas of pancreas and lungs , 22. In ogenesis , 24, 34.ironment .κB and both Ki-67 and p53 in the metastatic lymph nodes.The present study also revealed that Ki-67 expression was related to the size of the primary tumor and the number of metastatic lymph nodes. A significant positive correlation was found between NF-κB signal pathway, in cancer cells so far. In the present study, NF-κB, as a transcription factor involved in many physiological and pathological processes, was shown to play a role in CRC progression. It is of particular importance that NF-κB expression was correlated with poor prognostic factors including p53 and Ki-67. At present, many targeted molecules, such as Bortezomib which increases apoptosis by reducing NF-κB levels and decreases resistance to chemotherapy, are under research for the treatment of CRC [κB, which inhibits nuclear translocation, can be developed. As in the present study, revealing changes in NF-κB in primary and metastatic tumor cells in various types of cancers may accelerate the development of drugs against this molecule.Many potential target molecules have been studied to determine whether they could inhibit NF-t of CRC , 36. In CRC development is a multifactorial and multistep process. Activation of many oncogenes and inactivation of tumor suppressor genes play roles in this process. Many genetic and epigenetic changes are incorporated in the processes of normal colorectal mucosal epithelial transformation into carcinoma and tumor progression. CRC, with the exception of some well-defined familial syndromes, is mostly sporadic. Approximately 90% of CRC cases are thought to develop from colorectal adenomas with classic adenoma-carcinoma sequences . Changesp53 is a molecule in cell replication that arrests the cell cycle when there is DNA damage and when the damage cannot be repaired it leads to apoptosis. As a result of the mutation of p53, cells with damaged DNAs are likely to lead to cancer. Despite thousands of studies in literature, critical mechanisms in which p53 has been involved in carcinogenesis have not yet been fully explained. Mutated p53 has been observed in approximately 50% of all cancer cases and 60% of CRC cases, such that it is called “the guardian of the genome” . MutatedIt is known that an increase in proliferation/apoptosis rate characterizes oncogenesis due to the proliferation of the damaged cells that lose their apoptosis functions as a result of the p53 mutation. In this study, a higher Ki-67 proliferation rate in metastatic lymph nodes than the primary tumor cells may represent the increased mitotic capacity of metastatic cells in CRC.κB is a well-known transcription factor that has a role in development and progression of many cancers. NF-κB is claimed to have a role in apoptosis, angiogenesis, and proliferation effects and development of cancer. Higher expressions of NF-κB in adenomas than that of normal mucosa have been previously stated in colorectal cancer development [κB, like p53 and Ki-67, has been found to have a higher expression in metastatic lymph nodes than primary tumors in CRC patients.In addition to playing an important role in inflammation and immune responses, NF-elopment , 41, 42.κB can have a role in the development of CRC lymph node metastasis. These results showed that tumor cells in CRC lymph node metastasis may display a different biological behavior as compared to that of the primary tumor cells. In CRC patients, adjuvant chemotherapy applications affect the prognosis in lymph node metastases and in view of these results, more aggressive primary tumor cells with increased proliferation and decreased apoptosis rates seem to have caused the lymph node metastases.In CRC, many factors may have a role in tumor progression. According to the results of our study, p53 and NF-In recent years, with the integration of targeted treatments better clinical results are obtained in some selected cancer patients . In our κB activity has a unique role in the course of CRC. Increased NF-κB, p53, and Ki-67 expressions in metastatic tumors may have negative effects on either the natural clinical course or the response to treatments. Unfortunately, the data on microsatellite instability and KRAS mutations are (mostly) not available since this is a retrospective study. These markers would be useful since the presence of such lesions may interact with several pathways such as p53 mutations and also clinical outcomes. The finding that metastatic tumor cells in the lymph nodes have biological features differing from those of the primary tumor cells is particularly important in development of future treatment alternatives, such as NF-κB inhibitors. The results of this study also indicate that in CRC progression many other markers' expressions need to be researched.In view of the results of this study, it seems that NF-


The Editorial on the Research TopicHuman Tumor-Derived p53 Mutants: A Growing Family of Oncoproteinsp53 gene are collectively the most common event in human cancers. These do not merely reflect a loss of the tumor suppressive function of wild type (wt) p53 but are also selected during tumorigenesis for their acquired gain-of-function (GOF), together contributing to multiple hallmarks of cancer. Over 30 years of extensive study into wt p53 provided a wealth of information about its regulation, functions, and contribution to cancer prevention. Albeit, with a significant delay, the interest in mutant p53 has been growing fast over the past decade with the realization that most cancer patients present tumors with mutant p53, and these particularly manifest in aggressive and metastatic diseases. The growing understanding of mutant p53 exposes attractive therapeutic opportunities with wide clinical applications, which coincidently raise many challenging questions concerning associated complexities. In this research topic, we assembled 12 reviews exposing some critical issues and discussing prospect development in this field.Mutations in the tumor suppressor Kim et al.). The Lozano group also emphasized the biological and biochemical differences between different mutants, even between different substitutions of the same amino acid, such as p53R172H versus p53R172P. Hence, not all p53 mutants are equal. While the abovementioned mouse models for inherited p53 mutations (the Li–Fraumeni model), this represents a small fraction of p53 mutations in human cancers. This key point was discussed by the Lozano group highlighting the limitations of the current mouse models for sporadic p53 mutations in cancer. They discuss the problems with the current conditional mutant p53 models in which all cells are heterozygous for p53 from conception and hence do not faithfully mimic the role of mutant p53 in sporadic tumor development. These models lack the challenging context of the cells with wt p53 that normally comprise the tumor microenvironment and its inherent immune cells. There is a clear need for more sophisticated mouse models to better define the distinct roles of mutant p53 in these compartments. The Del Sal group  discussed the difference between the mouse and human mutant p53 exemplified by the p53R249S mutation, which exhibits GOF in human cells, but this had not been recapitulated in the relevant mouse model. A comprehensive list summarizing studies from the last decade is provided in the Del Sal review outlining which mutants GOF effects have been validated and the associated information.One of the most commonly used mouse models for cancer is the p53 knockout mouse. However, this model of p53 deficiency does not represent the majority of human cancers. A major leap in the understanding of mutant p53 regulation and GOF was derived from mouse genetics , 2. The Sabapathy discussed this important point in detail, emphasizing the timing and conditions under which dominant negative (DN) effects of mutant p53 occur, and when and how this would impact on tumorigenesis. His conclusion from the literature is that stress, whether acute  or chronic (activated oncogene), accumulates mutant p53; however, it is under the latter conditions that mutant p53 promotes tumorigenesis. Further complexity to this is the tissue specificity of the DN effect as learned from the KI heterozygote mouse models (Sabapathy). In the wake of the DN effect, there is often a loss of heterozygosity (LOH) of the wt allele. However, Walerych et al. discussed that LOH is also tissue specific, as exemplified by the work of Rotter and coworkers demonstrating that, in the embryonic stem cells of the KI mutant p53 mice, the LOH can be of either wt or mutant p53 alleles, potentially acting to control cell fate checkpoint (Sabapathy).Mutant p53 GOF requires the accumulation of the mutant protein and that, at least initially, it acts dominantly over the wt protein. Vijayakumaran et al. summarizes the differences and similarities in the regulation of wt and mutant p53. While both wt and mutant p53 are inherently labile proteins and accumulate in response to stress, only the mutant form remains stable. Intriguingly, wt and mutant p53 share many of their regulatory mechanisms. However, the loss of the key negative autoregulatory loops due to mutation in p53 result in the sustained accumulation of mutant p53 following stress conditions or exposure to oncogenic stress in cancer cells. This, together with a loss of specificity of additional E3 ligases toward mutant p53, provides an explanation for the accumulation of mutant p53. The additional complexity of p53 regulation, both wt and mutant p53, by microRNA (miRNA) is presented Vijayakumaran et al. This reveals the ways by which p53 can be deregulated in cancer but, at the same time, may define potential new therapeutic targets.A major requirement for GOF by mutant p53 is a constant stabilization of the mutant p53 protein, unlike the temporal accumulation of wt p53. The review by Giorgi et al. discussed the cytoplasmic apoptotic activities of wt p53, and the loss of these activities by mutations in p53. To date, there is no evidence for a GOF of mutant p53 directly regulating these activities . On the other hand, it was reported that mutant p53 proteins can aberrantly cooperate with known transcription factors by leading to disregulated gene expression. This results in increased proliferation, invasion, genomic instability, and chemoresistance. The interaction of mutant p53 with p53 family members p63 and p73 is key to some of its GOF. Ferraiuolo et al. review the intricate relationship between mutant p53 and p63 or p73. Mutant p53 proteins can also hamper tumor suppression transcriptional programs by binding to and displacing the p53 family members p73 and p63 from their consensus of target gene promoters . How are these plethora of activities achieved by mutant p53? At least two major mechanisms have been reviewed in this series. First, is by controlling gene expression through the alteration of specificity of certain transcription factors. Second, is by affecting chromatin remodeling through SWI/SNF and MLLs/MOZ . The effect of mutant p53 on MLLs/MOZ is achieved through ETS2, as reviewed in detail by Martinez. He discusses the mechanism by which mutant p53 protects ETS2 from degradation, and how this, in turn, affects the overall transcriptional effects of the ETS family and contributes to the oncogenic phenotype of mutant p53, such as increased nucleotide metabolic genes (Martinez). Bruno et al. reviewed the relationship of p53 with the cofactor Che-1/AATF. This provides an interesting example of a factor that acts as an activator and protector of both wt and mutant p53. In response to DNA damage, Che-1 induces the expression of wt and mutant p53, but activates wt p53 to induce growth arrest genes. In the case of mutant p53, it induces its expression and consequently the sequestration of p73 from apoptotic target genes, hence promotes survival .Beyond p53 family members, Haupt et al. reviewed the major tumor suppressive pathways, which are subverted by mutant p53, including PTEN, PLK2, and PML, which control the cell cycle and the latter also the circadian clock. Intriguingly, mutant p53 deregulates cellular metabolism including glucose, lipid, and nucleotide metabolism, ensuring the sufficient supply of building blocks to support tumor growth ; compounds that deplete mutant p53 expression, where HSP90, in particular Ganetespib, is the most advanced drug currently in phase III clinical trial; and explore other approaches, which are currently used for other oncogenes, including knockdown and read-through of premature termination . This review was complemented by two focused reviews on mutant p53 therapeutics. The first by Bykov et al., which focused on the mechanism of action by APR-246, including the refolding of mutant p53, the impact on mutant isoforms of p53 family members p63 and p73 and the effect on the cellular redox regulators, primarily glutathione and thioredoxin, to enhance oxidative stress. The potential of APR-246 as a single agent and in combination with DNA damaging agents is discussed, and the current clinical status of APR-246 and prospects are outlined . The second therapeutic review by Burgess et al. focused on MDM2/MDMX targeted therapies. This review provides a thorough overview of the current drugs and approaches to target p53 via MDM2 and MDMX pathways. They outline the clinical development of current MDM2 targeting compounds. Importantly, they discuss the major hurdle in this approach, which is severe cytopenias. Although, this approach has not been designed to target mutant p53, the relevance of mutant p53 to this therapeutic approach and the availability of appropriate biomarkers were discussed .A major consequence of mutant p53 GOFs is the acquired dependence of cancer cells on the expression of mutant p53. This dependence, which has been termed oncogenic addiction to mutant p53 has been discussed by multiple contributors to this series, highlighting its importance. Evidence for this addiction has been discussed by the Lozano, Sabapathy, and Del Sal groups. This addiction defines an Achilles Heal with important clinical implications. Overall, this series of reviews on mutant p53 expose the pivotal role of mutant p53 as an oncogenic driver and outline the fast advancement in our understanding of its regulation and oncogenic activities. Our deeper understanding of mutant p53 also highlights clear limitations, such as the differences between mutants p53 proteins and between mouse and human mutant p53. The lack of appropriate mouse models for somatic p53 mutations, which represents the most common event in human cancer is a major hurdle to our understanding of mutant p53 to the cancer cell versus the microenvironment. The series also reflects the excitement around the clinical opportunities and current clinical development but highlights the need of potent molecules to specifically target mutant p53, given its prevalence in human cancers. It has also been increasingly clear that mutant p53 proteins are not a single entity, but they behave as a family of oncoproteins whose deciphering and therapeutic tackling might impact enormously on the success of threatening the vast majority of human cancers.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Patients harbouring this genetic anomaly are commonly resistant to standard therapy. Thus, various p53 reactivating agents have been developed in order to restore its tumour suppressive abilities. Small molecular compounds, especially, have gained popularity in its efficacy against myeloma cells. For instance, promising preclinical results have steered both nutlin-3 and PRIMA-1 into phase I/II clinical trials. This review summarizes different modes of p53 inactivation in myeloma and highlights the current p53-based therapies that are being utilized in the clinic. Finally, we discuss the potential and promise that the novel small molecules possess for clinical application in improving the treatment outcome of myeloma. TP53 encodes for p53 tumor suppressor protein. Deemed the guardian of the genome, p53 safeguards the integrity of the genome and ensures that the tissue homeostasis is kept in check. Under normal physiological conditions, cellular p53 is expressed at low levels, thereby turning off the activity of p53 network. With stress induction, p53 is stabilized by means of posttranslational modifications, such as phosphorylation and acetylation [Located at the chromosome 17p13.1,tylation . The subtylation .Thirty years of intensive research on p53 have yielded significant understanding of its structure and basic functions. The high percentage of patients with p53 germline mutations succumbing to a wide range of cancer in Li-Fraumeni syndrome , 2, 4, 5In MM, mutation of p53 gene is a rare occurrence at diagnosis; however, the incidence increases as the stage of disease advances, suggesting its essential role in disease progression –8. OveraDeletion of chromosome 17p13 region, which contains the p53 gene locus, is a recurrent cytogenetic abnormality in MM and has been associated with less favorable outcome –11. p53 Fifty percent of cancer harbours p53 mutations, while in the remaining 50%, the wild type p53 is deemed to lose its function via various mechanisms that affect the expression and activity of p53. The main inhibition mechanism of p53 has been described to be the amplification or overexpression of its negative regulator mouse double minute 2 homolog (MDM2). MDM2 is an E3 ubiquitin ligase which promotes proteasomal degradation of p53 as well as inhibiting the transactivation domain of the tumor suppressor protein –23. UndeMDM4, a homolog of MDM2, does not have E3 ubiquitin ligase activity but inactivates p53 by binding to and inhibiting the transactivation domain of p53. Due to its essential role in inactivating p53, MDM4 dysregulation in cancer has also been receiving important attention lately , 25. In  TP53 is also a subject of intense research of late. Deregulation of miRNAs in cancer is being rigorously explored and this has led to the hypothesis of the role of this group of noncoding genes in the pathogenesis of MM [On top of that, epigenetic regulation ofis of MM , 31. miRis of MM , 33. Impis of MM . Introduis of MM .In addition, epigenetic factors are also possible regulators of the expression and the activity of p53. For instance, the deregulation of p14ARF has been reported to be responsible in abolishing the integrity of the p53 pathway . ARF has TP53 gene itself has also been demonstrated in human myeloma cell lines [Hypermethylation of the promoter region ofll lines , 40. Revll lines , 40.Collectively, these findings describe the diverse mechanisms of p53 inactivation in multiple myeloma.Due to the fact that p53 is the nexus of various tumor suppressive pathways, it is imperative to study the means of reactivating or restoring p53 functions in human cancer in order to revert or rescue cells from resistance towards standard chemotherapeutic treatments. In fact, many anticancer drugs induce apoptosis through multiple pathways that are at least in part dependent upon functional p53 activation. In the late 1980s and early 1990s, the introduction of wild type p53 gene into a variety of human tumor cells has shown induction of efficient growth inhibition and apoptosis . In lineIn myeloma, a preclinical study demonstrated that adenovirus mediated delivery of wild type p53 could potently induce apoptosis in myeloma cells while sparing the normal hematopoietic cells and normal lymphocytes . FurtherThe use of conventional or high-dose chemotherapy has been a long-standing approach to treat MM patients. First line therapy for eligible patients remains autologous stem cell transplantation following high-dose chemotherapy. This is often preceded by induction therapy to decrease tumor load by utilizing a combination of treatments that often include chemotherapeutic agents such as alkylating compounds . In relaBortezomib was approved by the Food and Drug Administration (FDA) for MM treatment in 2008 . BortezoDespite the introduction of bortezomib shifting the paradigm of MM treatment, the disease remains incurable and resistance towards this remarkable drug still arises. Given this situation, novel therapeutics targeting the critical p53 pathway is of utmost importance in order to reactivate the tumor suppressor network to execute apoptosis, in the hope of improving the treatment outcome in MM. In view of this, novel p53-reactivating agents have been developed and these agents are relevant to the nature and pathology of MM. The following describes these p53-reactivating drugs and the potential they hold for clinical applications. Due to the fact that newly diagnosed cases of MM are often presented with wild type p53, therapeutic induction of p53 is an attractive potential treatment strategy for this disease. The conventional way of inactivating wild type p53 is through deregulation/overexpression of MDM2, which inhibits the transcriptional activity of p53 as well as increasing the rate of the p53 degradation. In view of this, the development of drugs to reactivate wild type p53 has focused on developing small molecule inhibitors to the MDM2-p53 complex.(i) Nutlin. The first reported and the most well studied MDM2 inhibitor is the nutlins [ nutlins , 41. Nut nutlins . Nutlin  nutlins . In othe TP53 are potential attractive targets for MDM2 inhibitor-based therapy. Nutlin-3 has been shown to be a potent inducer of apoptosis in cell lines deriving from hematological malignancies, including MM, ALL, AML, CLL, and Hodgkin's lymphoma [Because MDM2 inhibitors depend on p53 activation in cells expressing wild type p53, hematological malignancies that mostly retain wild type genotype oflymphoma , 58. In lymphoma . It was lymphoma . This eflymphoma . The mollymphoma . ImperatFurthermore, nutlin displayed wide synergy with various conventional chemotherapeutic drugs, namely, melphalan, etoposide, and velcade , 60, 61.As mentioned earlier, monoallelic deletion of p53 with the remaining allele being a wild type is a recurrent cytogenetic abnormality in MM patients and this group of patients often suffers from poor prognosis. It still remains inconclusive whether the single allele of wild type p53 in MM cells is actually still functional or is able to be reactivated by a nongenotoxic agent such as nutlin-3. Our lab has shown that, in WT/- cases with high p53 expression, nutlin-3 was able to induce a functional p53 pathway, but with a compromised activity compared to the WT/WT cells, whereas in WT/- cases with low or zero p53 expression, nutlin-3 showed no efficacy. The findings indicate a haploinsufficient activity of p53 in myeloma .The current evidence holds a lot of promise for nutlin-3 to be translated into the clinic as a treatment for MM with wild type p53. However, it must be kept in mind that sporadic mutations of p53 could arise from selective pressure in the cells upon prolonged nutlin treatment, rendering a state of resistance towards the drug . Further(ii) RITA (Reactivation of p53 and Induction of Tumor Cell Apoptosis). RITA is a small molecule compound identified in a cell-based screen. It has a reversed mechanism from nutlin, whereby RITA binds to the amino terminal on p53 domain instead of on MDM2 protein. This binding causes conformational changes of p53 that reduces the p53-MDM2 interaction and hence decreases p53 ubiquitination which then leads to p53 accumulation, MDM2 downregulation, and p53-dependent apoptotic pathway induction [ in vitro findings, mouse xenograft models of MM which were subjected to RITA treatment displayed tumor regression and lengthened survival [nduction . Antimyesurvival .The efficiency of RITA as an antimyeloma agent was further strengthened when RITA was found to be able to overcome resistance of MM cells towards MDM2 inhibitors such as nutlin-3 and MI-63 . In thisThe clinical translation of RITA as an antimyeloma agent was further highlighted by its synergistic relationship with nutlin in inhibiting the growth and killing of MM cells .(iii) Other Small Molecules Inhibitors of p53-MDM2. The importance of MDM2 inhibitors in hematological malignancies was emphasized when these compounds, MI-63, MI-219, and MI-319, showed preclinical efficacies [ in vitro and in vivo evidence of reactivation of the p53 pathway [ficacies . Like nuficacies , 68. Theficacies . On the  pathway . These r(i) PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis). p53 mutations are often associated with resistance to chemotherapy treatment in cancer [n cancer , 15. Then cancer . This con cancer . Since m in vivo level, whereby it potently inhibits the growth of tumor in human tumor xenograft model [PRIMA-1 has been shown to have good efficacy against various types of solid cancer cells, namely, breast cancer , small cft model , 76. PRIft model –79. Due ft model .The potency of PRIMA-1 in hematological malignancies came to light when it was found to have an antileukemic effect in CLL and AML cells , 81. Int(ii) MIRA-1. MIRA-1, structurally distinct from PRIMA-1, is a maleimide compound that targets mutant p53 with higher potency than PRIMA-1 [ PRIMA-1 . MIRA-1  PRIMA-1 . First i PRIMA-1 .Since drug resistance is ubiquitous in multiple myeloma, drug combination has been employed as a treatment regime to improve the treatment outcome. For instance, nutlin-3 has been shown to act in concert with various conventional chemotherapeutics to activate p53, in efforts to improve the treatment efficacy and reduce the collateral damage caused by chemotherapy. Nutlin-3 was reported to induce growth arrest in normal cells, and upon removal of the drug, cell cycle resumes . As chem in vitro and in vivo antimyeloma effects, promising results arising from drug combinations suggest that combined use is a more attractive therapeutic option. The fact that p53-independent activities are involved in the drug mechanisms denotes that further investigations in this aspect are strongly called for so that we could fully utilize the versatility of the drug while maximizing its killing capacity. Of course, understanding the precise mechanism of action of these drugs would also aid in the future design of a novel and improved compound for the treatment of multiple myeloma.As p53 is the bridging point of apoptotic mechanisms, reactivation of the p53 pathway itself confers an excellent therapeutic approach in treating cancer. The current perspective points towards the importance of utilizing small molecular agents to reactivate the wild type p53 or to restore the transcriptional activities of the mutant p53. Even though each agent on its own was reported to show potent


Drug reaction with eosinophilia and systemic symptoms (DRESS) is a rare and severe adverse drug reaction. No pathognomonic test is available. The skin is the most frequent involved organ and easily sampled for a histopathological analysis. Recent series suggest that keratinocyte damage could be associated with either severe hepatic or renal involvement.To study the histopathologic findings in a large homogenous series of DRESS and their link, if there is any, with visceral involvement and severity.We describe in a retrospective large cohort, between 2005 and 2013, 45 patients diagnosed with a DRESS  and their cutaneous histological features.A facial edema in DRESS was associated with a definite case of DRESS (p=0.005) and a liver involvement (p=0.045). A younger age (50 years vs 66 years) was associated with a liver involvement (p=0.013). A severe neutropenia was associated with severe DRESS (p=0.012). Spongiosis and keratinocyte damage are the 2 most frequent epidermal changes noticed 55% and 53%. Pustules are not rare (26%). In the dermis a vascular alterations were frequent (88%) with a true leukocytoclastic vasculitis in 28%, a mild lymphocytic vasculitis in 13% and minor alterations in 44% of cases. In the dermis, there was a constant lymphocytic infiltrate mainly moderate in a perivascular distribution. In 57% the infiltrate is polymorphous with association of eosinophils neutrophil or atypical lymphocyte. In two third of cases, the changes are polymorphous and numerous, with no specific clear pattern. In the other one third of cases with few alterations, the presence of eosinophils, keratinocyte damage and spongiosis are usually present. A spongiosis was associated with non severe form of DRESS (p=0.041) and the absence of kidney involvement (p=0.023). A a higher of alterations per slides was associated a liver involvement (p=0.018). Our study do not support a prognostic value of keratinocyte damage.Histopathology of DRESS associated frequent spongiosis, keratinocyte damage, vascular alteration and polymorphism of the infiltrate. In two third of cases, the changes are polymorphous and numerous, in the other one third of cases with few alterations, the presence of eosinophils, keratinocyte damage and spongiosis are usually present. So cutaneous histopathology seems of little value to predict the visceral involvement.


Conformational and functional flexibility promote protein evolvability. High evolvability allows related proteins to functionally diverge and perhaps to neostructuralize. p53 is a multifunctional protein frequently referred to as the Guardian of the Genome–a hub for e.g. incoming and outgoing signals in apoptosis and DNA repair. p53 has been found to be structurally disordered, an extreme form of conformational flexibility. Here, p53, and its paralogs p63 and p73, were studied for further insights into the evolutionary dynamics of structural disorder, secondary structure, and phosphorylation. This study is focused on the post gene duplication phase for the p53 family in vertebrates, but also visits the origin of the protein family and the early domain loss and gain events. Functional divergence, measured by rapid evolutionary dynamics of protein domains, structural properties, and phosphorylation propensity, is inferred across vertebrate p53 proteins, in p63 and p73 from fish, and between the three paralogs. In particular, structurally disordered regions are redistributed among paralogs, but within clades redistribution of structural disorder also appears to be an ongoing process. Despite its deemed importance as the Guardian of the Genome, p53 is indeed a protein with high evolvability as seen not only in rearranged structural disorder, but also in fluctuating domain sequence signatures among lineages. Proteins are dynamic, with a natural tendency to rearrange their conformational ensembles in response to the local environment . ConformRecognized as the Guardian of the Genome, yet infamous for its frequent implication in cancer; p53 is a versatile protein, known to perform numerous functions from DNA binding as a transcription factor to a regulator of apoptosis and beyond . With poFor every amino acid substitution, the conformational and functional ensemble may be altered, with plausible scenarios ranging from no change to gain-or-loss of function. While globular protein domains must fold to function, structurally disordered regions may be less constrained, challenging the common concept of structure being more conserved than sequence. Many possibilities to balance the fitness equation exist if some functions are benefitted and others slightly impaired. This could result in an expanded nearly-neutral network that would allow rapid sequence divergence . HoweverL. gigantea that comprises the four domain cassette, fall inside the hemichordate and early chordate group. B. floridae has two copies; one (XP_002598770) has the p53 DBD and OD and falls far from all vertebrate p53 domains in this phylogeny, the other (XP_002613954) has the entire four domain cassette. This four domain cassette protein forms the closest outgroup to the entire vertebrate p53 family in this phylogeny and is considered the last common ancestor of all p53, p63 and p73 proteins in vertebrates, in agreement with taxonomy and previous studies [Reported sightings of a p53 protein and perhaps even a p63/p73 protein in choanoflagellates and invertebrates, suggest that the evolutionary record of p53 predates the beginning of the animal lineage, Metazoa . Thus, ar XP_0026954 has t studies . In vertB. floridae but before sharks diverged [B. floridae, all vertebrate proteins in the p53 family have lost domains, but no domains have been added. Proteins in the p63 and p73 clades overall share the three domain composition of p53 DBD, OD, and SAM. TAD is not identified by Pfam (P. sinensis and B. mutus) or p73 (U. maritimus), and OD is not found in two sequences in the p53 clade. One is after a lineage-specific duplication in E. edwardii and the second is from the only bird representative found in data derived from bird genome data, P. humilis. Lastly, the N-terminus and linkers between domains are variable in length, and in some cases linkers are even absent.The gene duplication pattern resulting in three vertebrate proteins from one ancestral protein is consistent with two whole genome duplications that supposedly occurred at the time of early vertebrates, after the divergence of diverged . To furtdiverged , but the by Pfam . In the  by Pfam . For theG. gallus [P. humilis [G. gallus p53 has the p53 DBD and the OD but like many other reptiles, it lacks TAD. Further, these two bird sequences fall outside the reptilian clade as the outgroup to mammals and thus, we cannot conclude that these are the main p53 proteins in P. humilis or G. gallus. However, given that G. gallus and P. humilis are distantly related birds and that they fall close to their expected location in the p53 family phylogeny  between human and shark sequences in the p53, p63, and p73 clades respectively reveal 51.55%, 76.13% and 76.65% sequence identity. Consequently, p63 and p73 are more similar, with 61.81% sequence identity when comparing shark sequences and 59.24% sequence identity when comparing the human sequences. Further, pairwise sequence identity for shark p53 vs. shark p63 and shark p73, reveal 49.02% and 49.86% respectively. Interestingly, the same comparisons made with the human proteins, p53 vs. p63 and p73, reveal 40.99% and 42.82% pairwise sequence identity, respectively. In summary, the shark p53 family proteins have diverged less than the human counterparts, in accordance with the significantly slower divergence rate found in sharks compared to other vertebrates .L. chalumnae have the most ordered DBD among the entire vertebrate p53 family  substitution rates per site (SEQ) were inferred . For allWith a high degree and varying amount of disorder across the p53 family, an analysis of the secondary structure elements propensities was suitable. Mapped in a heat map context, similar to that for the disorder propensity, reveal multiple regions with secondary structure transitions between sequences in the same clade and in a clade-specific manner . To quanSince phosphorylation frequently modulates the conformations of disordered regions in a regulatory fashion, an analysis of predicted phosphorylation sites was conducted. Here the heat map shows the locations of predicted phosphorylation sites in a binary fashion. Since only Ser, Thr, and Tyr can be phosphorylated, the amount of Ser, Thr, and Tyr may also be important for how many phosphorylation sites are predicted. However, while there are significant differences in the fraction of Ser, Thr, and Tyr among the different clades  there is no significant difference in the fraction of sites predicted to be phosphorylated when comparing p53, p63 and p73 mean values . In all clades, about 5% of all sites are predicted to be phosphorylated . To quanRegions that are rapidly changing in disorder, secondary structure, and phosphorylation are likely less important for a conserved function. These rates are calculated for the entire vertebrate p53 family and clade-specific patterns are therefore indistinct. To gain resolution on the clade level, clade-specific rates were estimated  Figs. PlAll prediction methods applied are intentionally based on linear sequences and not on 3D structures since the repertoire of 3D structures, although quite impressive for the p53 family, may only provide a limited set of snapshots of the conformational ensemble in which these proteins exist. However, the structural context is valuable and site specific DOT as well as the site specific fractions of predicted disorder were mapped onto structures for TAD, p53 DBD, and OD  .For TAD, the MDM2 binding site is shown . Here, mFor p53 DBD, the tetrameric state with DNA bound is displayed for the p53 family,  but for For OD, the two different tetrameric states are displayed for the p53 family,  but for To investigate if phosphorylation may be one of the mechanisms utilized to differentiate the regulatory pathways of p53, p63, and p73 from each other, shared and clade-specific phosphorylation sites were identified using a 50% majority rule either within a clade or across the entire p53 family. In total, 66 phosphorylation sites were identified . Of thesUsing linear sequence predictors, properties of structural disorder (IUPred), secondary structure (PSIPRED), and phosphorylation sites (NetPhos) have been inferred. It is important to remember that these are predictions and cannot be perfect given that they are (i) independently aiming to predict traits that may depend on each other, (ii) using only the linear sequence context without considering long-range sequence contacts, and (iii) based on experimental data that may not reflect the dynamic nature of a protein sequence, e.g. one PDB structure is merely a snapshot of a conformational ensemble . The accTrichoplax adhaerens, have an MDM2 binding site [Caenorhabditis elegans [B. floridae . Both oInevitably, ongoing functional divergence is present in the p53 family, and especially in the p53 clade. The Guardian of the Genome gives the impression of still exploring its function and does not fit the picture of a resilient Guardian. Perhaps, a more appropriate way to refer to p53 is as a Gambler of the Genome?Three datasets were constructed: (i) the p53 protein family at the whole protein level in vertebrates, (ii) the p53 protein family at the nucleotide level in vertebrates, and (iii) the p53 protein family at the DNA-binding domain level in a representative set of vertebrate sequences and non-vertebrates. For (i), NCBI BLAST  was perfHomo sapiens, Bos taurus, Gallus gallus, Anolis carolinensis, Xenopus tropicalis, Latimeria chalumnae, Takifugu rubripes, Danio rerio, and Callorhinchus milii. Sequence identifiers for all vertebrate sequences are given in For major vertebrate taxonomic groups, a representative organism with sequence information for all three paralogs in the p53 protein family was selected from (i). Vertebrate organisms included in (iii) were: Monosiga brevicollis and Salpingoeca rosetta. The resulting topology was used to guide rooting the trees from the first two datasets by rooting on the branch containing both p63 and p73 clades and selecting the p53 clade as the outgroup.Sequences for datasets (i) and (iii) were aligned with MAFFT v7.123–1  using thTo assess the characterization of the structural properties of the proteins included in our phylogenies, the amino acid sequence of each protein  was used as input for different sequence-based predictors in order to predict structural disorder, secondary structure, phosphorylation sites and domain regions. Thereafter, for each prediction method, the predicted value for each residue in each protein sequence was mapped onto its corresponding site in the multiple sequence alignment. This resulted in three matrices for (i) structural disorder prediction, (ii) secondary structure predictions, and (iii) predicted phosphorylation sites. For (i) and (iii), the data predicted was continuous. For (ii), the data had three non-numerical categories. In order to analyze the transitions between order and disorder, between the presence of secondary structure elements and loops, and for presence or absence of phosphorylation sites, all matrices were represented as binary phyletic patterns (as described below). The phyletic patterns were individually analyzed in their phylogenetic context and transition rates were calculated.Structural disorder was predicted using IUPred ,62 versiSecondary structure was predicted using PSIPRED ,64 versiPhosphorylation sites for Serine, Threonine and Tyrosine residues were predicted using NetPhos  version Protein domains were predicted based on Pfam  version Rate4Site  was usedTo study the gain/loss transitions of structural properties in related proteins along their evolutionary history, a protocol that includes the estimation of evolutionary rates per site based on the phylogenetic trees and the binary matrices generated was adopted. GLOOME software  was usedInference methods implemented in R statistical software  were useConservation, here defined as the fraction of disorder per site from the binary matrices (gaps included), was calculated for the p53 family and the individual clades. Site specific rates and conservation of disorder were mapped onto representative PDB structures for the different domains. S1 FigOverview of the p53 family phylogeny including 74 representative species across Metazoa and Choanoflagellates, built based on their p53 DBD domains. Support values at the nodes indicate posterior probabilities. Nodes with posterior probability < 0.5 are unresolved.(PDF)Click here for additional data file.S2 Fighttp://tree.bio.ed.ac.uk/software/figtree/).(A) Circular representations of p53 DNA-based phylogeny and (B) its corresponding full-protein-based phylogeny. These consensus trees were obtained with MrBayes 3.2.2 after sampling trees for 15 million generations with the default burn-in phase (discarding the first 25% of trees) and using the 50% majority rule. Node circles show posterior probabilities ranging from 0.5 in red to 1 in white. Here proteins were colored by clade  with tip labels following the color guide from (PDF)Click here for additional data file.S3 Fig(A) Heat map showing Pfam domain predictions per protein into their corresponding multiple sequence alignment sites , all in the context of the p53 DNA-based phylogeny with tip labels colored according to the color guide. (B) In addition, individual domain architectures (labeled and colored as shown in Pfam domains box) were also included to highlight their actual lengths enforcing missing or broken domains. Figure generated with iTOL .(PDF)Click here for additional data file.S4 FigDistribution of structural disorder (grey) and order (blue) in full-length proteins and in p53 DBD domains sorted by p53 DNA-based phylogenetic tree with tip labels following the color guide. Furthermore, individual domain architectures (labeled and colored as shown in Pfam domains box) were also included. Figure generated with iTOL .(PDF)Click here for additional data file.S5 Fig2 = 0.41, and p-value < 0.05, concluding that linear correlation different to 0 is statistically significant).Scatter plot of the p53 DBD structural disorder percentage vs. the number of Pfam domains per protein from 74 hits, including invertebrates and vertebrates proteins. There is a positive correlation between these two variables Click here for additional data file.S6 FigBoxplots showing the fractions of serine, threonine and tyrosine residues per protein per clade compared to the fractions of sites predicted to be phosphorylated by NetPhos  using a (PDF)Click here for additional data file.S7 FigCombined profiles of normalized evolutionary rates per aligned site for family and clades (vertebrates set) comparing disorder-order transitions (DOT) with (A) amino acid substitutions (SEQ), (B) secondary structure elements-loop transitions (SLT), and (C) phosphorylation transitions (PT). Grey shaded areas delimitate Pfam domain regions.(PDF)Click here for additional data file.S8 FigCombined profiles of normalized evolutionary rates per aligned site for family and clades (vertebrates set) comparing amino acid substitutions (SEQ) with (A) secondary structure elements-loop transitions (SLT) and (B) phosphorylation transitions (PT). Grey shaded areas delimitate Pfam  domain r(PDF)Click here for additional data file.S9 FigHeat map for structural traits plotted in the order of the p53 DNA-based phylogenetic tree context, showing p53 protein names as boxes colored according to the color guide in (PDF)Click here for additional data file.S1 TableAccession numbers for the vertebrate datasets (i) and (ii).(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableAlignment sites following a 50% majority rule of sequences with phosphorylation predictions based on NetPhos phosphorylation prediction score cut-off = 0.75 (gaps included). Information displayed per clade (specific) and per family (shared). Shaded areas correspond to the majority rule (phosphorylation predicted for more than 50% of taxa per clade or for the family). Corresponding positions in the canonical human proteins  are shown.(PDF)Click here for additional data file.