Expression of Wild-Type p53 by Curcumin, Alpinetin and Flavokawain B in Colorectal Cancer cells Expressing R273H Mutant p53

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

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High p53 Protein Expression LEVEL Independent Of Mutational Status Is An Adverse Prognostic Factor For Survival In ACUTE Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1330.1330 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1330-1330

Author(s):

Alfonso Quintas-Cardama ◽

Sean M. Post ◽

Kensuke Kojima ◽

Yi Hua Qiu ◽

Michael Andreeff ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mutant P53 ◽

P53 Mutations ◽

Wild Type ◽

P53 Pathway ◽

P53 Expression ◽

Mutational Status ◽

Wild Type P53 ◽

Acute Myeloid

Abstract Background The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML), particularly in cases with high-risk cytogenetics. It has been shown that p53 stabilization, which frequently occurs when the protein is mutated, can compromise its function. We have shown that p53 stabilization, regardless of the presence of mutations, suggesting alterations of other components in the p53 pathway. Methodology p53 expression was determined using high-throughput reverse phase protein array (RPPA) technology in 719 samples from 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status of p53 alleles was assessed by Sanger sequencing of exons 5 through 9. Expression of components of the p53 pathway was determined using standard immunohistochemical techniques. Nutlin-3a was used in in vitro culture experiments. Results Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). p53 expression correlated directly with CD34 (p=0.001) and inversely correlated with WBC (p=0.007), PB and BM blast burden (p=0.0001), and survival (p=0.01). High p53 (p53high) expression was more associated with unfavorable cytogenetics, particularly -5 (p=0.00001). p53high resulted in lower complete remission (CR) rates (51% vs 56%; p=??), higher relapsed rates (82% vs 62%; p=??), and shorter median overall survival (OS; 29.8 vs. 51 wks, p=0.009) compared to p53low pts. Most cases with p53high had unfavorable cytogenetics. We next correlated p53 stabilization with the presence of p53 mutations in 68 pts. p53 mutations were detected in 20/54 (37%) p53high pts and in 0/14 (0%) pts with p53low. p53high, either in the presence (29 wks) or in the absence (24 wks) of p53 mutations (p=1.0), was associated with significantly shorter OS compared with p53low pts (56 wks; p=0.05). Multivariate analysis revealed p53 expression to be an independent risk factor for survival in AML (p=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), BAD (p=0.0001), cleaved PARP (p=0.002), and cleaved PARP (p=0.01), and negatively with p21 (p=0.01), and MDM2 (p=0.001).Given the similar OS in p53high pts carrying mutant or wild-type p53, we scored the immunohistochemical expression of MDM2, MDM4, and p21 in 30 p53high pts (9 p53 mutated, 21 wild-type p53). Overexpression of MDM2 was observed in 44% vs 48% pts with mutant vs wild-type p53, respectively, whereas rates were 67% vs 62% for MDM4, and 0% vs 19% for p21, for each respective genotype. Overall, of the 21 p53high pts carrying wild-type p53, 15 (71%) had overexpression of MDM2 and/or MDM4, whereas 81% had no p21 expression, indicating deficient activation of the p53 pathway similar to those cases carrying mutant p53. We are currently assessing response to nutlin-3a therapy in 24 primary AML samples (4 mutant p53, 20 wild-type p53). Results showing the impact of p53 mutation and/or stabilization, and expression levels of MDM2, MDM4, and p21 on nutlin-3a therapy will be presented. Conclusions p53 stabilization (p53high) is a powerful predictive and prognostic factor in AML, which is independent of the presence of mutant p53 alleles. Poor outcomes in pts with p53high lacking p53 mutations are very frequently associated with overexpression of negative regulators of p53 such as MDM2 and/or MDM4 and p21 downregulation, indicating a functionally altered p53 pathway. These findings may have implications for therapies targeting the MDM2/p53 axis in AML. Disclosures: No relevant conflicts of interest to declare.

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Mutant p53 elicits context-dependent pro-tumorigenic phenotypes

Oncogene ◽

10.1038/s41388-021-01903-5 ◽

2021 ◽

Author(s):

Jennifer J. McCann ◽

Irina A. Vasilevskaya ◽

Christopher McNair ◽

Peter Gallagher ◽

Neermala Poudel Neupane ◽

...

Keyword(s):

Prostate Cancer ◽

Tumor Suppressor ◽

Dna Binding ◽

Mutant P53 ◽

Wild Type ◽

P53 Expression ◽

Transcriptional Profiles ◽

Cancer Types ◽

Context Dependent ◽

Wild Type P53

AbstractThe tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular “hot spots” in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

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Targeting Aggregation of Wilde-Type p53 and Mutant p53 with ReACp53 As a Novel Therapeutic Concept for AML

Blood ◽

10.1182/blood.v128.22.3944.3944 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3944-3944 ◽

Cited By ~ 1

Author(s):

Jianfang Zeng ◽

Alice Soragni ◽

Jo Ishizawa ◽

Vivian Ruvolo ◽

Christopher B. Benton ◽

...

Keyword(s):

Cell Lines ◽

Term Treatment ◽

Mutant P53 ◽

Amyloid Aggregation ◽

Wild Type ◽

Short Term ◽

Short Term Treatment ◽

P53 Expression ◽

Hematopoietic Malignancies ◽

Wild Type P53

Abstract Background: The tumor suppressor p53 is a master regulator of apoptosis, autophagy, cell cycle, and senescence. It is inactivated via mutation in approximately 50% of solid tumors, but only in 15% of hematopoietic malignancies including acute myeloid leukemia (AML). A recently proposed mechanism has linked loss of p53 function with its amyloid aggregation. Conceptually, certain p53 mutations can favor partial unfolding of the protein and expose a natively buried aggregation-prone segment. This can result in amyloidogenic aggregation and prevent p53 transcriptional activity and anti-tumor functions. The cell-permeable peptide ReACp53, has been recently developed to block p53 aggregation and restore its transcriptional function in the nucleus as well as its ubiquitination by MDM2. ReACp53 showed significant cytotoxicity in ovarian cancer but no toxicity to normal hematopoietic cells in animal experiment. We sought to determine the anti-tumor activity of ReACp53 in hematopoietic malignancies. Results: We examined the p53 status in 23 malignant hematopoietic cell lines by PCR, Sanger sequencing, and immunoblotting. Two cell lines were null for p53 expression, one harbored frame shift mutations, 11 cell lines expressed various missense p53 mutations, one cell line had an in frame deletion of p53, and eight cell lines expressed wild-type p53. Additionally, immunofluorescence staining (IF) with the conformation-specific PAb240 antibody revealed high levels of cytoplasmic, partially unfolded p53 in the cells expressing mutant p53. In p53 wild-type cells, p53 protein was mainly localized in the nucleus and was negative for PAb240. The p53 null and frame shift-mutant cells showed no p53 expression. All the cells were treated short-term with various concentrations of ReACp53, or a scrambled peptide, and assessed for apoptosis by flow cytometry. We found that ReACp53 was cytotoxic not only to the p53-mutant cells, but also to the wild-type p53 lines. In fact, all p53 wild type AML cell lines were highly sensitive. The p53 negative cell lines were seemingly resistant to short-term exposure to ReACp53. DeltaNp73, an isoform of p73 that antagonizes p53 and TAp73, is expressed in most AML cells and also has a similar aggregation-prone segment. We examined the levels of DeltaNp73 and total p73 in 12 AML cell lines by PCR, immunoblotting, and IF. Both proteins were overexpressed in all five wild-type p53 cell lines, and DeltaNp73 was predominately localized in the cytoplasm of these cells. After short-term treatment with ReACp53, DeltaNp73 expression and localization didn't change in wild type p53 AML cells. Over-expressing DeltaNp73 in HEK293T cells enhanced their level of Thioflavin T staining indicating amyloid aggregation of the protein. Compared to controls, the DeltaNp73 overexpressing HEK293T cells were more prone to apoptosis following ReACp53 treatment. Absent of transactivation domain, DeltaNp73 is not expected to be restored to function like TAp73. Mutant p53 is known to cross-aggregate p73 and p63 because of their highly similar aggregation-prone segments, therefore, we hypothesize that DeltaNp73 cross-aggregated p53 and p73 and ReACp53 inhibited the aggregation as to restore p53 and TAp73 function and exposure to MDM2. We chose two wild-type p53 AML cell lines, OCI-AML3 and MOLM-14, which express MDM2 and are sensitive to the MDM2 inhibitor DS3032b. After short-term treatment with ReACp53, p53 and p73 (also a MDM2 target) expression decreased significantly in both cells. We tested the anti-leukemia efficacy of the DS3032b and ReACp53 combinatorial treatment in these cells and found that DS3032b synergized with ReACp53 to efficiently kill the cells compared to the cytotoxic activity of DS3032b or ReACp53 treatment alone. Conclusions: We demonstrate a new mechanism of DeltaNp73 inhibition of wild-type p53 and TAp73 mediated by induction of amyloid aggregation. ReACp53 showed apoptogenic efficacy in malignant hematopoietic cells, both in cells expressing wild-type p53 as well as mutant p53. In the wild-type AML cells where p73 and DeltaNp73 were overexpressed, sensitivity to ReACp53 increased. ReACp53 also exhibited synergistic activity when combined with the MDM2 inhibitor DS3032b in wild-type p53 cells. Together, our data suggest a novel mechanism of p53 inactivation by amyloid formation, that can be corrected in acute myeloid leukemia carrying either wild-type or mutant p53. Disclosures No relevant conflicts of interest to declare.

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Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5581-5592.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5581-5592 ◽

Cited By ~ 3

Author(s):

E Shaulian ◽

A Zauberman ◽

D Ginsberg ◽

M Oren

Keyword(s):

Dna Binding ◽

Point Mutation ◽

P53 Gene ◽

Dominant Negative ◽

Mutant P53 ◽

Wild Type ◽

Transforming Activity ◽

Wild Type P53 ◽

Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

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Mutant p53 as an Antigen in Cancer Immunotherapy

International Journal of Molecular Sciences ◽

10.3390/ijms21114087 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4087 ◽

Cited By ~ 1

Author(s):

Navid Sobhani ◽

Alberto D’Angelo ◽

Xu Wang ◽

Ken H. Young ◽

Daniele Generali ◽

...

Keyword(s):

Cancer Cells ◽

Cancer Patients ◽

Current Knowledge ◽

Adaptive Immune Response ◽

Mutant P53 ◽

Wild Type ◽

P53 Antibodies ◽

Wild Type P53

The p53 tumor suppressor plays a pivotal role in cancer and infectious disease. Many oncology treatments are now calling on immunotherapy approaches, and scores of studies have investigated the role of p53 antibodies in cancer diagnosis and therapy. This review summarizes the current knowledge from the preliminary evidence that suggests a potential role of p53 as an antigen in the adaptive immune response and as a key monitor of the innate immune system, thereby speculating on the idea that mutant p53 antigens serve as a druggable targets in immunotherapy. Except in a few cases, the vast majority of published work on p53 antibodies in cancer patients use wild-type p53 as the antigen to detect these antibodies and it is unclear whether they can recognize p53 mutants carried by cancer patients at all. We envision that an antibody targeting a specific mutant p53 will be effective therapeutically against a cancer carrying the exact same mutant p53. To corroborate such a possibility, a recent study showed that a T cell receptor-like (TCLR) antibody, initially made for a wild-type antigen, was capable of discriminating between mutant p53 and wild-type p53, specifically killing more cancer cells expressing mutant p53 than wild-type p53 in vitro and inhibiting the tumour growth of mice injected with mutant p53 cancer cells than mice with wild-type p53 cancer cells. Thus, novel antibodies targeting mutant p53, but not the wild-type isoform, should be pursued in preclinical and clinical studies.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.12 ◽

1991 ◽

Vol 11 (1) ◽

pp. 12-19 ◽

Cited By ~ 132

Author(s):

J Milner ◽

E A Medcalf ◽

A C Cook

Keyword(s):

Tumor Progression ◽

Carboxyl Terminus ◽

Mutant P53 ◽

Tumor Suppressor P53 ◽

Wild Type ◽

Dominant Effect ◽

Type Mutant ◽

Wild Type P53

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.

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Analysis of HPV16 E6 and mutant p53-transfected keratinocytes in reconstituted epidermis suggests that wild-type p53 inhibits cytokeratin 19 expression

Journal of Cell Science ◽

10.1242/jcs.107.2.435 ◽

1994 ◽

Vol 107 (2) ◽

pp. 435-441

Author(s):

J.P. Moles ◽

J.T. Schiller ◽

A. Tesniere ◽

I.M. Leigh ◽

J.J. Guilhou ◽

...

Keyword(s):

Human Keratinocytes ◽

Genetic Alterations ◽

Cytokeratin 19 ◽

Mutant P53 ◽

Wild Type ◽

Early Passage ◽

Hpv16 E6 ◽

Ck19 Expression ◽

Wild Type P53

Using a reconstituted skin culture model we have analysed the effects of oncogenic human papillomavirus (HPV) and mutant TP53 genes on the proliferation and differentiation of human keratinocytes. Immortal cell lines generated by transfection of early passage normal human keratinocytes with HPV16 E7 plus mutant human TP53 (KN #1), HPV16 E7/E6 (KN #2), or HPV16 E7 plus murine p53 (KN #3) were examined. KN #1 and KN #2 behaved identically, reconstructing a tumor-like epidermis characterized by the lack of differentiation and the presence of an aberrant epidermal architecture. In contrast, KN #3 reconstructed an epidermis that was more similar to that obtained with normal keratinocytes. KN #1 and KN #2 were further characterized by the inversion of the proliferative compartment and the abnormal expression of cytokeratin 19 (CK19). Because p53 function is reduced in these cells, either by heterocomplex formation between endogenous wild-type p53 and transfected mutant p53 or by E6-induced degradation of wild-type p53, we hypothesized that CK19 expression may be normally repressed by wild-type p53. This hypothesis was supported by the strict correlation observed between TP53 mutation and CK19 expression in a set of human skin tumors. CK19 was detected in all eight carcinomas containing a mutated TP53 gene but in none of the 16 carcinomas containing only wild-type TP53. These results illustrate the utility of the in vitro reconstituted skin model for investigating the consequences of genetic alterations in human keratinocytes.

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Resuscitating Wild-Type p53 Expression by Disrupting Ceramide Glycosylation: A Novel Approach to Target Mutant p53 Tumors: Figure 1.

Cancer Research ◽

10.1158/0008-5472.can-11-0700 ◽

2011 ◽

Vol 71 (20) ◽

pp. 6295-6299 ◽

Cited By ~ 18

Author(s):

Yong-Yu Liu

Keyword(s):

Mutant P53 ◽

Wild Type ◽

P53 Expression ◽

Novel Approach ◽

Wild Type P53

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.12-19.1991 ◽

1991 ◽

Vol 11 (1) ◽

pp. 12-19 ◽

Cited By ~ 1

Author(s):

J Milner ◽

E A Medcalf ◽

A C Cook

Keyword(s):

Tumor Progression ◽

Carboxyl Terminus ◽

Mutant P53 ◽

Tumor Suppressor P53 ◽

Wild Type ◽

Dominant Effect ◽

Type Mutant ◽

Wild Type P53

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.

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