Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.     

The transforming and suppressor functions of p53 alleles: effects of mutations that disrupt phosphorylation, oligomerization and nuclear translocation.

Mutant p53 alleles that have a recessive phenotype in human tumors can, in cooperation with an activated H-ras gene, transform rat embryo fibroblasts (REFs). Mutant p53 proteins differ from wild type, and from each other in conformation, localization and transforming potential. Missense mutations in codons 143, 175 and 275 confer strong transforming potential. A serine 135 p53 mutant has an intermediate transforming potential, while the histidine codon 273 allele transforms weakly, if at all. In contrast to the wild type p53 gene, mutant p53 alleles with strong transforming ability cannot suppress the transformation of REFs by other oncogenes. The His273 allele retains partial suppressor function in this assay. The relevance of p53 oligomerization, phosphorylation and nuclear translocation to the transforming potential of mutant p53 and to wild type p53 suppressor function were examined. The inability of mutant p53 polypeptides to form homodimers correlates with loss of transforming function. Monomeric variants of wild type p53 protein, however, retain the ability to suppress focus formation. Phosphorylation of serine residues 315 and 392 is not required for the transforming function of mutant p53, nor is serine 315 required for suppressor function when these alleles are constitutively expressed in REF assays. Nuclear translocation-defective mutant and wild type p53 proteins retain transforming and suppressor function in REF assays.     

Mutant p53 protein is targeted by arsenic for degradation and plays a role in arsenic-mediated growth suppression

p53 is frequently mutated in tumor cells, and mutant p53 is often highly expressed due to its increased half-life. Thus, targeting mutant p53 for degradation might be explored as a therapeutic strategy to manage tumors that are addicted to mutant p53 for survival. Arsenic trioxide, a drug for patients with acute promyelocytic leukemia, is found to target and degrade a class of proteins with high levels of cysteine residues and vicinal thiol groups, such as promyelocytic leukemia protein (PML) and PML-retinoic acid receptor α fusion protein. Interestingly, wild type p53 is accumulated in cells treated with arsenic compounds, presumably due to arsenic-induced oxidative stresses. In this study, we found that wild type p53 is induced by arsenic trioxide in tumor cells, consistent with published studies. In contrast, we found that arsenic compounds degrade both endogenous and ectopically expressed mutant p53 in time- and dose-dependent manners. We also found that arsenic trioxide decreases the stability of mutant p53 protein through a proteasomal pathway, and blockage of mutant p53 nuclear export can alleviate the arsenic-induced mutant p53 degradation. Furthermore, we found that knockdown of endogenous mutant p53 sensitizes, whereas ectopic expression of mutant p53 desensitizes, tumor cells to arsenic treatment. Taken together, we found that mutant p53 is a target of arsenic compounds, which provides an insight into exploring arsenic compound-based therapy for tumors harboring a mutant p53.     

Inability of p53-reactivating compounds Nutlin-3 and RITA to overcome p53 resistance in tumor cells deficient in p53Ser46 phosphorylation

The p53 tumor suppressor protein plays key roles in protecting cells from tumorigenesis. Phosphorylation of p53 at Ser46 (p53Ser46) is considered to be a crucial modification regulating p53-mediated apoptosis. Because the activity of p53 is impaired in most human cancers, restoration of wild-type p53 (wt-p53) function by its gene transfer or by p53-reactivating small molecules has been extensively investigated. The p53-reactivating compounds Nutlin-3 and RITA activate p53 in the absence of genotoxic stress by antagonizing the action of its negative regulator Mdm2. Although controversial, Nutlin-3 was shown to induce p53-mediated apoptosis in a manner independent of p53 phosphorylation. Recently, RITA was shown to induce apoptosis by promoting p53Ser46 phosphorylation. Here we examined whether Nutlin-3 or RITA can overcome resistance to p53-mediated apoptosis in p53-resistant tumor cell lines lacking the ability to phosphorylate p53Ser46. We show that Nutlin-3 did not rescue the apoptotic defect of a Ser46 phosphorylation-defective p53 mutant in p53-sensitive tumor cells, and that RITA neither restored p53Ser46 phosphorylation nor induced apoptosis in p53Ser46 phosphorylation-deficient cells retaining wt-p53. Furthermore, treatment with Nutlin-3 or RITA together with adenoviral p53 gene transfer also failed to induce apoptosis in p53Ser46 phosphorylation-deficient cells either expressing or lacking wt-p53. These results indicate that neither Nutlin-3 nor RITA in able to induce p53-mediated apoptosis in the absence of p53Ser46 phosphorylation. Thus, the dysregulation of this phosphorylation in tumor cells may be a critical factor that limits the efficacy of these p53-based cancer therapies.     

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

Mutant p53 reactivation by small molecules makes its way to the clinic

The TP53 tumor suppressor gene is mutated in many human tumors, including common types of cancer such as colon and ovarian cancer. This illustrates the key role of p53 as trigger of cell cycle arrest or cell death upon oncogenic stress. Most TP53 mutations are missense mutations that result in single amino acid substitutions in p53 and expression of high levels of dysfunctional p53 protein. Restoration of wild type p53 function in such tumor cells will induce robust cell death and allow efficient eradication of the tumor. Therapeutic targeting of mutant p53 in tumors is a rapidly developing field at the forefront of translational cancer research. Various approaches have led to the identification of small molecules that can rescue mutant p53. These include compounds that target specific p53 mutations, including PK083 and PK5174 (Y220C mutant p53) and NSC319726 (R175H mutant p53), as well as PRIMA-1 and its analog APR-246 that affect a wider range of mutant p53 proteins. APR-246 has been tested in a Phase I/II clinical trial with promising results.     

Mutant p53: it’s not all one and the same

Mutation of the TP53 tumor suppressor gene is the most common genetic alteration in cancer, and almost 1000 alleles have been identified in human tumors. While virtually all TP53 mutations are thought to compromise wild type p53 activity, the prevalence and recurrence of missense TP53 alleles has motivated countless research studies aimed at understanding the function of the resulting mutant p53 protein. The data from these studies support three distinct, but perhaps not necessarily mutually exclusive, mechanisms for how different p53 mutants impact cancer: first, they lose the ability to execute wild type p53 functions to varying degrees; second, they act as a dominant negative (DN) inhibitor of wild type p53 tumor-suppressive programs; and third, they may gain oncogenic functions that go beyond mere p53 inactivation. Of these possibilities, the gain of function (GOF) hypothesis is the most controversial, in part due to the dizzying array of biological functions that have been attributed to different mutant p53 proteins. Herein we discuss the current state of understanding of TP53 allele variation in cancer and recent reports that both support and challenge the p53 GOF model. In these studies and others, researchers are turning to more systematic approaches to profile TP53 mutations, which may ultimately determine once and for all how different TP53 mutations act as cancer drivers and whether tumors harboring distinct mutations are phenotypically unique. From a clinical perspective, such information could lead to new therapeutic approaches targeting the effects of different TP53 alleles and/or better sub-stratification of patients harboring TP53 mutant cancers.Subject terms: Cancer genetics, Tumour-suppressor proteins     

The aggregation of mutant p53 produces prion-like properties in cancer

The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.     

Biogenesis of p53 involves cotranslational dimerization of monomers and posttranslational dimerization of dimers. Implications on the dominant negative effect.

Precisely how mutant p53 exerts a dominant negative effect over wild type p53 has been an enigma. To understand how wild type and mutant p53 form hetero-oligomers, we studied p53 biogenesis in vitro. We show here that p53 dimers are formed cotranslationally (on the polysome), whereas tetramers are formed posttranslationally (by the dimerization of dimers in solution). Coexpression of wild type and mutant p53 therefore results in 50% of the p53 generated being heterotetramers comprised of a single species: wild type dimer/mutant dimer. Using hot spot mutants of p53 and a variety of natural target sites, we show that all wild type/mutant heterotetramers manifest impaired DNA binding activity. This impairment is not due to the mutant dimeric subunit inhibiting association of the complex with DNA but rather due to the lack of significant contribution (positive cooperativity) from the mutant partner. For all heterotetramers, bias in binding is particularly pronounced against those sequences in genes responsible for apoptosis rather than cell growth arrest. These results explain the molecular basis of p53 dominant negative effect and suggest a functional role in the regulation of p53 tetramerization.     

Dissecting the pathways that destabilize mutant p53: The proteasome or autophagy?

One fundamental feature of mutant forms of p53 consists in their accumulation at high levels in tumors. At least in the case of neomorphic p53 mutations, which acquire oncogenic activity, stabilization is a driving force for tumor progression. It is well documented that p53 mutants are resistant to proteasome-dependent degradation compared with wild-type p53, but the exact identity of the pathways that affect mutant p53 stability is still debated. We have recently shown that macroautophagy (autophagy) provides a route for p53 mutant degradation during restriction of glucose. Here we further show that in basal conditions of growth, inhibition of autophagy with chemical inhibitors or by downregulation of the essential autophagic genes ATG1/Ulk1, Beclin-1 or ATG5, results in p53 mutant stabilization. Conversely, overexpression of Beclin-1 or ATG1/Ulk1 leads to p53 mutant depletion. Furthermore, we found that in many cell lines, prolonged inhibition of the proteasome does not stabilize mutant p53 but leads to its autophagic-mediated degradation. Therefore, we conclude that autophagy is a key mechanism for regulating the stability of several p53 mutants. We discuss plausible mechanisms involved in this newly identified degradation pathway as well as the possible role played by autophagy during tumor evolution driven by mutant p53.     

Tumorigenic p53 mutants undergo common structural disruptions including conversion to α‐sheet structure

The p53 protein is a commonly studied cancer target because of its role in tumor suppression. Unfortunately, it is susceptible to mutation‐associated loss of function; approximately 50% of cancers are associated with mutations to p53, the majority of which are located in the central DNA‐binding domain. Here, we report molecular dynamics simulations of wild‐type (WT) p53 and 20 different mutants, including a stabilized pseudo‐WT mutant. Our findings indicate that p53 mutants tend to exacerbate latent structural‐disruption tendencies, or vulnerabilities, already present in the WT protein, suggesting that it may be possible to develop cancer therapies by targeting a relatively small set of structural‐disruption motifs rather than a multitude of effects specific to each mutant. In addition, α‐sheet secondary structure formed in almost all of the proteins. α‐Sheet has been hypothesized and recently demonstrated to play a role in amyloidogenesis, and its presence in the reported p53 simulations coincides with the recent re‐consideration of cancer as an amyloid disease.