p53 mutants without a functional tetramerisation domain are not oncogenic

p53 is altered in about 50 % of cancers. Most of the p53 mutants have lost the wild-type tumour suppressor activity but show oncogenic properties. The majority of the p53 alterations are missense mutations of residues located in its DNA binding domain (DBD). Only a few mutations concern residues in its tetramerisation domain (TD). However, the study of mutant proteins identified in tumors that do not form tetramers has shown that they have lost the wild-type activity like most of the p53 DBD mutants. Here, we show that two of such mutant proteins, Arg342Pro and Leu344Pro are not dominant negative and do not stimulate the expression of a reporter gene under the control of the multi-drug resistance gene-1 (MDR-1). This suggests that to be oncogenic, p53 mutants need to form tetramers. Accordingly, the dominant negative effect and the ability of a tetrameric mutant protein, Asp281Gly, to stimulate the MDR-1 promoter are abolished when its TD is rendered non-functional by the mutation of leucine 344 to a proline residue. These results suggest that mutations in the TD, are less selected in tumors than mutations in the DBD because they do not lead to oncogenic proteins.     

Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis

Wild‐type p53 functions as a tumour suppressor while mutant p53 possesses oncogenic potential. Until now it remains unclear how a single mutation can transform p53 into a functionally distinct gene harbouring a new set of original cellular roles. Here we show that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated full‐length p53 mRNA. Cells expressing mutant p53 exhibit “gain‐of‐function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Interestingly, Δ160p53‐overexpressing cells behave in a similar manner. In contrast, an exogenous or endogenous mutant p53 that fails to express Δ160p53 due to specific mutations or antisense knock‐down loses pro‐oncogenic potential. Our data support a model in which “gain‐of‐function” phenotypes induced by p53 mutations depend on the shorter p53 isoforms. As a conserved wild‐type isoform, Δ160p53 has evolved during millions of years. We thus provide a rational explanation for the origin of the tumour‐promoting functions of p53 mutations.     

Estimating the selective advantage of mutant p53 tumour cells to repeated rounds of hypoxia

The tumour suppressor gene, p53, plays an important role in tumour development. Under low levels of oxygen (hypoxia), cells expressing wild-type p53 undergo programmed cell death (apoptosis), whereas cells expressing mutations in the p53 gene may survive and express angiogenic growth factors that stimulate tumour vascularization. Given that cells expressing mutations in the p53 gene have been observed in many forms of human tumour, it is important to understand how both wild-type and mutant cells react to hypoxic conditions. In this paper a mathematical model is presented to investigate the effects of alternating periods of hypoxia and normoxia (normal oxygen levels) on a population of wild-type and mutant p53 tumour cells. The model consists of three coupled ordinary differential equations that describe the densities of the two cell types and the oxygen concentration and, as such, may describe the growth of avascular tumours in vitro and/or in vivo. Numerical and analytical techniques are used to determine how changes in the system parameters influence the time at which mutant cells become dominant within the population. A feedback mechanism, which switches off the oxygen supply when the total cell density exceeds a threshold value, is introduced into the model to investigate the impact that vessel collapse (and the associated hypoxia) has on the time at which the mutant cells become dominant within vascular tumours growing in vivo. Using the model we can predict the time it takes for a subpopulation of mutant p53 tumour cells to become the dominant population within either an avascular tumour or a localized region of a vascular tumour. Based on independent experimental results, our model suggests that the mutant population becomes dominant more quickly in vivo than in vitro (12 days vs 17 days).     

Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.     

A stable human p53 heterotetramer based on constructive charge interactions within the tetramerization domain

The human p53 tetramerization domain (called p53tet; residues 325-355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold.     

Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

Alterations in the tumour suppressorp53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of thep53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5–9 of thep53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13⋅6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients.     

HDAC inhibitors show differential epigenetic regulation and cell survival strategies on p53 mutant colon cancer cells

Besides inactivating tumour suppressor activity in cells, mutations in p53 confer significant oncogenic functions and promote metastasis and resistance to anticancer therapy. A variety of therapies involving genetic and epigenetic signalling events regulate tumorogenesis and progression in such cases. Pharmacological interventions with HDAC inhibitors have shown promise in therapy. This work explores the changes in efficacy of the four HDAC inhibitors SAHA, MS-275, valproic acid and sodium butyrate on a panel of colon cancer cell lines – HCT116 (p53 wt), HCT116 p53-/-, HT29 and SW480 (with mutations in p53). Clonogenic assays, gene profiling and epigenetic expression done on these cells point to p53 dependent differential activity of the 4 HDAC inhibitors which also elevate methylation levels in p53 mutant cell lines. In silico modelling establishes the alterations in interactions that lead to such differential activity of valproic acid, one of the inhibitors considered for the work. Molecular Dynamic simulations carried out on the valproic acid complex ensure stability of the complex. This work establishes a p53 dependent epigenetic signalling mechanism triggered by HDAC inhibition expanding the scope of HDAC inhibitors in adjuvant therapy for p53 mutant tumours.     

Crystal structure of a superstable mutant of human p53 core domain. Insights into the mechanism of rescuing oncogenic mutations

Most of the cancer-associated mutations in the tumor suppressor p53 map to its DNA-binding core domain. Many of them inactivate p53 by decreasing its thermodynamic stability. We have previously designed the superstable quadruple mutant M133L/V203A/N239Y/N268D containing the second-site suppressor mutations N239Y and N268D, which specifically restore activity and stability in several oncogenic mutants. Here we present the x-ray structure of this quadruple mutant at 1.9 A resolution, which was solved in a new crystal form in the absence of DNA. This structure reveals that the four point mutations cause only small local structural changes, whereas the overall structure of the central beta-sandwich and the DNA-binding surface is conserved. The suppressor mutation N268D results in an altered hydrogen bond pattern connecting strands S1 and S10, thus bridging the two sheets of the beta-sandwich scaffold in an energetically more favorable way. The second suppressor mutation N239Y, which is located in close proximity to the DNA-binding surface in loop L3, seems to reduce the plasticity of the structure in large parts of loop L3 as indicated by decreased crystallographic temperature factors. The same is observed for residues in the vicinity of the N268D substitution. This increase in rigidity provides the structural basis for the increase in thermostability and an understanding how N268D and N239Y rescue some of the common cancer mutants.     

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman,Atomic Force and Surface Plasmon Resonance spectroscopies

BackgroundMutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in > 50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding.MethodsMolecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28.ResultsWe show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge.ConclusionsThese results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function.General significanceRaman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.     

Regulating the genome surveillance system: miRNAs and the p53 super family

The p53 gene super family consists of three members; TP53, TP63 and TP73, encoding proteins p53, p63 and p73. Whilst p63 appears to have an essential role in embryonic development with a less clear role in carcinogenesis, irregularities in p53 and p73 signalling are implicated in tumour formation. As such, p53 is a tumour suppressor which is mutated in over 50% cancers and p73 was recently formally classified as a tumour suppressor based on data showing p73 deficient mice generate spontaneous tumours similar to those observed in p53 null mice. Dysregulation of both p53 and p73 has been correlated with cancer progression in many cell types and although mutation of these genes is often observed, some form of p53/p73 deregulation likely occurs in all tumour cells. The discovery that complementary micro RNAs (miRNAs) are able to target both of these genes provides a potential new means of perturbing p53/p73 signalling networks in cancer cells. Here we summarise the current literature regarding the involvement of miRNAs in the modulation of p53 family proteins and cancer development and detail the use of in silico methods to reveal key miRNA targets.     

SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.Methods and resultsBy cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.ConclusionsSLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.General SignificanceThis work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.     

Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.     

Loss of PML cooperates with mutant p53 to drive more aggressive cancers in a gender-dependent manner

p53 mutations and downregulation of promyelocytic leukemia (PML) are common genetic alterations in human cancers. In healthy cells these two key tumor suppressors exist in a positive regulatory loop, promoting cell death and cellular senescence. However, the influence of their interplay on tumorigenesis has not been explored directly in vivo. The contribution of PML to mutant p53 driven cancer was evaluated in a mouse model harboring a p53 mutation (p53^{wild-type/R172H}) that recapitulates a frequent p53 mutation (p53^R175H) in human sporadic and Li-Fraumeni cancers. These mice with PML displayed perturbation of the hematopoietic compartment, manifested either as lymphoma or extramedullary hematopoiesis (EMH). EMH was associated with peripheral blood leucocytosis and macrocytic anemia, suggestive of myeloproliferative- myelodysplastic overlap. In contrast, a complete loss of PML from these mice resulted in a marked alteration in tumor profile. While the incidence of lymphomas was unaltered, EMH was not detected and the majority of mice succumbed to sarcomas. Further, males lacking PML exhibited a high incidence of soft tissue sarcomas and reduced survival, while females largely developed osteosarcomas, without impact on survival. Together, these findings demonstrate that PML is an important tumor suppressor dictating disease development in a pertinent mouse model of human cancer.

Key Points: (1) A mutant p53 allele disrupts hematopoiesis in mice, by promoting lymphomas and myeloproliferative / myelodysplastic overlap. (2) Coincidental p53 allele mutation and PML loss shifts the tumor profile toward sarcoma formation, which is paralleled in human leiomyosarcomas (indicated by immunohistochemistry; IHC).