P53 Variants Research Articles

Δ122p53, a mouse model of Δ133p53α, enhances the tumor-suppressor activities of an attenuated p53 mutant.

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92–495 days) which was much greater than mΔpro/- mice (range 120–250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78–150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.

Structure of full-length p53 tumor suppressor probed by chemical cross-linking and mass spectrometry.

The tumor suppressor p53 presents a great challenge for 3D structural analysis due to its inherent flexibility. In this work, we gained insight into the structure of full-length wild-type human p53 in solution by chemical cross-linking/MS. This approach allowed us obtaining structural information of free wild-type p53 in solution without making use of the ultrastable quadruple p53 variant. The cross-links within one p53 monomer are in good agreement with the small-angle X-ray scattering based model of full-length p53. Our cross-linking data between different p53 molecules in the tetramer however indicate a large degree of flexibility in the C-terminal regulatory domain of full-length p53 in the absence of DNA. The cross-links suggest that the C-terminal regulatory domains are much closer to each other, resulting in a more compact arrangement of the p53 tetramer than perceived by the small-angle X-ray scattering model.

Highlights of This Issue

Alternative pre-mRNA splicing plays a critical role in cancer development, progression, and metastasis. To elucidate alternative splicing events associated with cancer metastasis, Lu and colleagues carried out comprehensive transcriptome analyses of cancer cells derived from in vitro and in vivo models of metastatic colonization. Deep RNA sequencing revealed an extensive splicing regulatory network involving multiple splicing factors and their downstream targets that affect the invasiveness and metastatic behavior of cancer cells. These findings underscore the importance of alternative splicing in cancer metastasis, and suggest possible molecular biomarkers and therapeutic targets.The proliferative heterogeneity within human tumors complicates the diagnosis and treatment of cancer patients because slow proliferators are hard to eradicate, can be difficult to detect, and may cause disease relapse sometimes years after apparently curative treatment. Here, Dey-Guha and colleagues identify a novel β1-integrin–associated signaling pathway that rapidly proliferating cancer cells occasionally trigger to divide asymmetrically and produce a slowly proliferating daughter cell. These findings reveal a targetable mechanism for reducing the proliferative heterogeneity within cancer cell populations and have broad implications for understanding cancer progression, dormancy, and therapeutic resistance.Over 30 years after the discovery of p53, the critical regulators of its “life versus death” decision still remain unclear. Whereas clear data indicate that a common polymorphism in p53 (proline 72 or arginine 72) influences this decision, the underlying basis is not understood. Kung and colleagues identify TRIML2 as a gene that is preferentially induced by the arginine 72 variant of p53. Furthermore, TRIML2 interacts with p53 and induces SUMOylation of this protein. This facilitates the ability of p53 to transactivate specific proapoptotic target genes, thus revealing a novel mediator of p53-mediated apoptosis.Annexin A2 (ANXA2) expression is high in normal prostate glands, becomes lost in low-grade tumors, but reemerges in advanced tumors. Examination of primary prostate cancer specimens by Griner and colleagues, using immunocytochemistry and gene expression analysis, revealed a reciprocal relationship of ANXA2 and ERG. In a majority of cases, ANXA2 was absent or reduced in well-differentiated ERG-positive tumors but was expressed at moderate to high levels in ERG-negative tumors with poorly differentiated features. This reciprocal association is demonstrated by mechanistic studies to be mediated by the transcriptional repression of ANXA2 by ERG and will be helpful for the stratification of patients.

Identification of TRIML2, a Novel p53 Target, that Enhances p53 SUMOylation and Regulates the Transactivation of Proapoptotic Genes

The tumor-suppressor protein p53, encoded by TP53, inhibits tumorigenesis by inducing cell-cycle arrest, senescence, and apoptosis. Several genetic polymorphisms exist in TP53, including a proline to arginine variant at amino acid 72 (P72 and R72, respectively); this polymorphism alters p53 function. In general, the P72 variant shows increased ability to induce cell-cycle arrest, whereas the R72 variant possesses increased ability to induce apoptosis, relative to P72. At present, the underlying mechanisms for these functional differences are not fully understood. Toward elucidating the molecular basis for these differences, a gene-expression microarray analysis was conducted on normal human fibroblast cells that are homozygous for P72 and R72 variants, along with subclones of these lines that express a p53 short hairpin (shp53). Approximately three dozen genes were identified whose transactivation is affected by the codon 72 polymorphism. One of these is the tripartite-motif family-like 2 (TRIML2) gene, which is preferentially induced by the R72 variant. Importantly, the accumulated data indicate that TRIML2 interacts with p53, and facilitates the modification of p53 with SUMO2. TRIML2 also enhances the ability of p53 to transactivate a subset of proapoptotic target genes associated with prolonged oxidative stress, including PIDD, PIG3 (TP53I3), and PIG6 (PRODH). These data indicate that TRIML2 is part of a feed-forward loop that activates p53 in cells expressing the R72 variant, particularly after prolonged stress. The defined actions of TRIML2, in part, explain the underlying molecular basis for increased apoptotic potential of the R72 variant of p53.

Guilty as CHARGED: p53's expanding role in disease

Unrestrained p53 activity during development, as occurs upon loss of the p53 negative regulators Mdm2 or Mdmx, causes early embryonic lethality. Surprisingly, co-expression of wild-type p53 and a transcriptionally-dead variant of p53, with mutations in both transactivation domains (p53L25Q,W26S,F53Q,F54S), also causes lethality, but later in gestation and in association with a host of very specific phenotypes reminiscent of a syndrome known as CHARGE. Molecular analyses revealed that wild-type p53 is inappropriately activated in p535,26,53,54/+ embryos, triggering cell-cycle arrest or apoptosis during development to cause CHARGE phenotypes. In addition, CHARGE syndrome is typically caused by mutations in the CHD7 chromatin remodeler, and we have shown that activated p53 contributes to phenotypes caused by CHD7-deficiency. Together, these studies provide new insight into CHARGE syndrome and expand our understanding of the role of p53 in diseases other than cancer.

A genetic variant of p53 restricts the mucous secretory phenotype by regulating SPDEF and Bcl-2 expression.

Despite implications for carcinogenesis and other chronic diseases, basic mechanisms of p53 and its variants in suppressing Bcl-2 levels, are poorly understood. Bcl-2 sustains mucous cell metaplasia, whereas p53−/− mice display chronically increased mucous cells. Here we show that p53 decreases bcl-2 mRNA half-life by interacting with the 5’ untranslated region (UTR). The p53-bcl-2 mRNA interaction is modified by the substitution of proline by arginine within the p53 proline-rich domain (PRD). Accordingly, more mucous cells are present in primary human airway cultures with p53Arg compared with p53Pro. Also, the p53Arg compared with p53Pro displays higher affinity to and activates the promoter region of SAM-pointed domain-containing Ets-like factor (SPDEF), a driver of mucous differentiation. On two genetic backgrounds, mice with targeted replacement of prolines in p53 PRD show enhanced expression of SPDEF and Bcl-2 and mucous cell metaplasia. Together, these studies define the PRD of p53 as a determinant for chronic mucus hypersecretion.

A balancing act: orchestrating amino-truncated and full-length p73 variants as decisive factors in cancer progression.

p73 is the older sibling of p53 and mimics most of its tumor-suppressor functions. Through alternative promoter usage and splicing, the TP73 gene generates more than two dozen isoforms of which N-terminal truncated DNp73 variants have a decisive role in cancer pathogenesis as they outweigh the positive effects of full-length TAp73 and p53 in acting as a barrier to tumor development. Beyond the prevailing view that DNp73 predominantly counteract cell cycle arrest and apoptosis, latest progress indicates that these isoforms acquire novel functions in epithelial-to-mesenchymal transition, metastasis and therapy resistance. New insight into the mechanisms underlying this behavior reinforced the expectation that DNp73 variants contribute to aggressive cellular traits through both loss of wild-type tumor-suppressor activity and gain-of-function, suggesting an equally important role in cancer progression as mutant p53. In this review, we describe the novel properties of DNp73 in the invasion metastasis cascade and outline the comprehensive p73 regulatome with an emphasis on molecular processes putting TAp73 out of action in advanced tumors. These intriguing insights provoke a new understanding of the acquisition of aggressive traits by cancer cells and may help to set novel therapies for a broad range of metastatic tumors.

EMT by p53ψ

A transcriptionally inactive p53 variant promotes mesenchymal phenotypes from the mitochondria.

Combined effects of leukocyte telomere length, p53 polymorphism and human papillomavirus infection on esophageal squamous cell carcinoma in a Han Chinese population

Telomere shortening has been suggested to be a genetic predictor for various cancers. However, evidences about this point with respect to esophageal squamous cell carcinoma (ESCC) in Han Chinese populations remain limited. Our previous study demonstrated that p53 Arg72Pro polymorphism was associated with the risk of human papillomavirus (HPV)-related ESCC. Telomeres and p53 play important roles in maintaining genomic stability and regulating the cell cycle. HPV impacts both telomere length stabilization and p53 degradation. Given the roles of the three factors, we evaluated leukocyte telomere length, p53 variants and HPV-16 serology to examine the potential associations between them and ESCC risk in a case–control study with 308 patients and 309 cancer-free controls matched by age and sex. Compared with long telomere length, short telomere length was significantly associated with an increased risk of ESCC (adjusted OR 2.01; 95% CI 1.41–2.80). Moreover, this association was enhanced when combined with HPV-16 seropositivity and p53 Arg/Arg or Arg/Pro genotypes. Notably, individuals with short telomere length, Arg/Pro or Arg/Arg genotypes and HPV-16 seropositivity had a 12.08-fold (95% CI 5.49–26.56) increased risk of ESCC compared to those with none of the three investigated risk factors. Taken together, these results indicate that short telomere length in peripheral blood leukocytes is a biomarker for ESCC risk, and has statistically additive effects with p53 variants and HPV seropositivity with regard to the risk of ESCC in a Han Chinese population.

Brazilian story of the R337H p53 mutation

The p53 tumor suppressor is an evergreen of molecular oncology. Since its discovery in 1979, it has been subjected to intensive investigation. The p53 protein is composed of "only" 393 amino acid residues, and function of almost each of them has been addressed in detail. Somatic mutations are extremely frequent, they can be found almost in each of the p53 codons and in all types of tumors. Inherited p53 mutations are rare but very penetrant, and they are typically associated with development of a broad spectrum of tumors. However, in 2001, the p53 research provided an unexpected discovery: the R337H allele was found in southern Brazil. This allele was atypically associated with only one type of tumor - childhood adrenocortical carcinoma and it exhibited low penetrance. Therefore, new data on functioning and impact of the R337H mutation were highly desired. The results obtained during a few following years helped to elucidate not only this specific p53 variant but also provided insight into general principles of mutant p53 variants function. It also turned out that all R337H alleles that are very frequent in southern Brazil originate from one common ancestor.

Inappropriate p53 activation during development induces features of CHARGE syndrome.

CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.

Discovery of a p53 variant that controls metastasis.

The tumor suppressor p53 encoded by the tumor protein 53 (TP53) gene has been extensively studied since its discovery in 1979 (1, 2). The last four decades have seen the publication of an enormous number of reports on the role of p53 in suppressing cancer formation. These studies have demonstrated that TP53 mutations are the most common genetic alterations in human tumors (3).

Re-engineered p53 activates apoptosis in vivo and causes primary tumor regression in a dominant negative breast cancer xenograft model.

Inactivation of p53 pathway is reported in more than half of all human tumors and can be correlated to malignant development. Missense mutation in the DNA binding region (DBD) of p53 is the most common mechanism of p53 inactivation in cancer cells. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain (TD). Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have previously designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain capable of escaping transdominant inhibition by mutant p53 in vitro. In this report, we demonstrate the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model in vivo. In addition, we illustrate the profound effects of the dominant negative effect of endogenous mutant p53 over wt-p53 in cancer cells. Finally, we investigate the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model.

Characterization of a new mouse p53 variant: loss-of-function and gain-of-function.

Backgroundp53 is a major tumor suppressor that is inactivated in over 50% of human cancer types through either mutation or inactivating interactions with viral or cellular proteins. The uncertainties around the link between p53 status, therapeutic response, and outcome in cancer suggest that additional factors may be involved. p53 isoforms that are generated via the alternative splicing pathway may be promising candidates for further investigation.ResultIn this study, we report one new p53 protein with two internally deleted regions, resulting in one deleted amino acid fragment (from amino acid residues 42 to 89) and one reading frame-shift region (from amino acid residues 90-120) compared to wild-type p53. The functional status of the new p53 protein, which has a defect in its proline-rich and N-terminal DNA-binding domains, was characterized as possessing an intact conformation, exhibiting no transactivation activity, exerting a dominant-negative effect and an interacting with a coactivator with an arginine methyltransferase activity.ConclusionTaken together, our findings provide valuable information about the structure and function of p53 for the regulation of transactivation activity and cellular protein-protein interactions. Furthermore, natural p53 isoforms will help us understand the functional roles of the p53 family and potential therapeutics for p53-dependent cancers.

P0104MDM2 and P53 codon 72 polymorphisms in acute myeloid leukaemia

Background Acute myeloid leukaemia (AML) is a heterogenous disease with numerous genetic abnormalities corresponding to a variety of subtypes. P53 is a principle mediator of multiple cellular functions including cell cycle arrest, senescence, and apoptosis in response to cellular stresses. The activity of P53 may either be inactivated or attenuated in a vast majority of human cancers through mutations in the P53 gene or aberrant expression of proteins acting in the P53 tumour suppression pathway, such as its key cellular regulator MDM2. Single nucleotide polymorphism (SNP) at codon 72 of the P53 gene (A-G exchange) alters the primary structure of the P53 protein. MDM2 SNP309 is a single nucleotide T to G polymorphism located in the promoter of the MDM2 gene which increase the expression of MDM2, thereby impairing the tumour suppressor activity of P53. We aimed at defining the role of MDM2 SNP309 and P53 codon 72 polymorphisms in the risk of AML. Methods Genotyping for MDM2 SNP309 and P53 codon 72 polymorphisms was done by AS-PCR and PCR-RFLP techniques respectively, for 50 de-novo adult AML patients and 50 healthy age and sex matched controls. Findings We found a borderline significant statistical difference between cases and controls regarding combined MDM2 T/G and P53 genotypes. There was no significant correlation between either MDM2 SNP309 or P53 codon 72 polymorphisms alone and the risk of AML. MDM2 variant genotypes were significantly associated with lower age of onset of AML and lower haemoglobin level while P53 variants were significantly associated with lower CD117 expression. Interpretation An interactive effect between MDM2 SNP309 and P53 codon 72 polymorphisms may increase the risk of AML.

The Dermal Layer of Sweet Sorghum (Sorghum bicolor) Stalk, a Byproduct of Biofuel Production and Source of Unique 3-Deoxyanthocyanidins, Has More Antiproliferative and Proapoptotic Activity than the Pith in p53 Variants of HCT116 and Colon Cancer Stem Cells

There is a growing interest in the utilization of sweet sorghum as a renewable resource for biofuels. During the biofuel production process, large quantities of biomass are generated, creating a rich source of bioactive compounds. However, knowledge of sweet sorghum stalk is lacking. We measured the phenolic content (Folin-Ciocalteu assay), antioxidant activity (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) assay), and phytochemical composition (LC-MS) in both the pith and dermal layer of the stalk. We further tested the antiproliferative (5-bromo-2'- deoxyuridine assay) and proapoptotic (terminal deoxynucleotidyl transferase dUTP nick end labeling assay) activities of these extracts using HCT116 cells and colon cancer stem cells (CCSCs) with and without the tumor suppressor gene p53. For the first time, we show that the dermal layer extract of sweet sorghum contains more of the 3-deoxyanthocyanidins apigeninidin and luteolinidin than the pith, and this is associated with more anticancer activity. Furthermore, luteolinidin suppressed CCSC proliferation more than apigeninidin. In addition to being renewable biofuel, sweet sorghum may also serve as a source of health-promoting compounds.

Phase I Study of Oral Rigosertib (ON 01910.Na), a Dual Inhibitor of the PI3K and Plk1 Pathways, in Adult Patients with Advanced Solid Malignancies

To determine the pharmacokinetics (PK), maximum tolerated dose (MTD), safety, and antitumor activity of an oral formulation of rigosertib, a dual phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathway inhibitor, in patients with advanced solid malignancies. Patients with advanced solid malignancies received rigosertib twice daily continuously in 21-day cycles. Doses were escalated until intolerable grade ≥2 toxicities, at which point the previous dose level was expanded to define the MTD. All patients were assessed for safety, PK, and response. Urinary PK were performed at the MTD. Archival tumors were assessed for potential molecular biomarkers with multiplex mutation testing. A subset of squamous cell carcinomas (SCC) underwent exome sequencing. Forty-eight patients received a median of 2 cycles of therapy at 5 dose levels. Rigosertib exposure increased with escalating doses. Dose-limiting toxicities were hematuria and dysuria. The most common grade ≥2 drug-related toxicities involved urothelial irritation. The MTD is 560 mg twice daily. Activity was seen in head and neck SCCs (1 complete response, 1 partial response) and stable disease for ≥12 weeks was observed in 8 additional patients. Tumors experiencing ≥partial response had PI3K pathway activation, inactivated p53, and unique variants in ROBO3 and FAT1, two genes interacting with the Wnt/β-catenin pathway. The recommended phase II dose of oral rigosertib is 560 mg twice daily given continuously. Urinary toxicity is the dose-limiting and most common toxicity. Alterations in PI3K, p53, and Wnt/β-catenin pathway signaling should be investigated as potential biomarkers of response in future trials.

Relationship among tobacco habits, human papilloma virus (HPV) infection, p53 polymorphism/mutation and the risk of oral squamous cell carcinoma.

The prevalence of oral squamous cell carcinoma (OSCC) has significantly increased over decades in several countries and human papilloma virus (HPV) has been indicated as one of the underlying causes. This suggests that HPV plays a role in the early stages of carcinogenesis but is not a requisite for the maintenance and progression of malignant state. p53 is a tumor suppressor gene that checks the cell and promotes apoptosis and cell repair that can be deactivated by mutations and a viral interaction leading to cancer and individuals with particular polymorphic variant of p53 is more susceptible to HPV-induced carcinogenesis. The present study has been carried out to detect and correlate p53 polymorphism/mutation, HPV DNA in the biopsy samples of oral cancer patients who had tobacco habits.

A genetic variant of p53 restricts the mucous secretory phenotype by regulating SPDEF and Bcl-2 expression

A genetic variant of p53 restricts the mucous secretory phenotype by regulating SPDEF and Bcl-2 expression

Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most intriguing findings is that mutant p53 appears as discrete dot-shaped regions within the nucleus of breast cancer cells. In many malignant cells, the nucleolar sequestration of p53 is evident. These observations support the view that the nucleolus is involved directly in the mediation of p53 function or indirectly by the control of the localization of p53 interplayers. p53 expressed in the nuclear fraction of breast cancer cells revealed a wide spectrum of isoforms. p53 isoforms ΔNp53 (47 kDa) and Δ133p53β (35 kDa), known as dominant-negative repressors of p53 function, were detected as the most predominant variants in nuclei of invasive breast carcinoma cells. The isoforms expressed also varied between individual tumors, indicating potential roles of these p53 variants in human breast cancer.