Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome

Nathaniel C. Holcomb; Amanda M. Harrington; Hong Pu; Berina Halilovic; Nathan R. Shelman; Shulin Zhang; Catherine Sears; Terra Armstrong; Brent Shelton; Lauren Corum; John A. D'Orazio

doi:10.1371/journal.pone.0320036

Abstract

We identified a germline TP53 c.758C > T (p.T253I) mutation in the TP53 tumor suppressor gene in a pediatric adrenocortical carcinoma (ACC) patient. Characteristic of pathogenic p53 mutations, we observed upregulation of total p53 protein levels in the patient’s ACC and concurrent suppression of the wild-type (WT) TP53 allele. As ACC can be associated with Li-Fraumeni Syndrome (LFS) and the mutation has not yet been linked to LFS, we sought to characterize the functionality of the T253I mutation. We acquired p53^-/- HEK293 cells and stably transduced them with GFP-tagged wild type (T253) or T253I p53 as well as two established pathogenic p53 mutants (C176Y and R213X). Compared to p53 WT, levels of T253I p53 increased while MDM2 levels decreased, suggesting a loss of MDM2-mediated regulation of T253I p53. Additionally, T253I showed a reduction in DNA damage responsive events, diminished DNA binding capabilities, and blunted transactivation capacity. These experimental data lead us to conclude that T253I represents a pathologic variant in TP53 that may predispose to LFS-associated tumors.

Citation: Holcomb NC, Harrington AM, Pu H, Halilovic B, Shelman NR, Zhang S, et al. (2025) Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome. PLoS One 20(12): e0320036. https://doi.org/10.1371/journal.pone.0320036

Editor: Swati Palit Deb, Virginia Commonwealth University, UNITED STATES OF AMERICA

Received: February 18, 2025; Accepted: October 30, 2025; Published: December 5, 2025

Copyright: © 2025 Holcomb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: We have made all data used in this manuscript publicly available at the online data repository FigShare with the following DOI: 10.6084/m9.figshare.27902106. The complete data set for each figure in the manuscript is available at the following link https://figshare.com/articles/dataset/Figure_1_A_and_B_-_Data_and_Analysis/27902106 You can also find this data set by searching the DOI listed above on DOI.org.

Funding: Kentucky Pediatric Cancer Research Trust Fund NCI P30 CA177558 NIH P20 GM121327.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome caused by an inherited defect in one allele of the TP53 tumor suppressor gene or similar loss of function of a gene in the p53 damage response pathway [1,2]. LFS significantly raises lifetime risk of a variety of pediatric and adult-type cancers including sarcomas, brain tumors, melanoma, breast cancer, adrenocortical carcinoma (ACC) and leukemia [3,4]. Penetrance of LFS is variable, but it is estimated that germline loss-of-function mutations in TP53 can be associated with up to a 70% lifetime risk of cancer in men and up to a 90% lifetime risk of cancer in women, with a large number of patients presenting with their first malignancy in childhood, adolescence and young adulthood [5,6]. It is now appreciated that LFS represents a spectrum of clinical phenotypic severity based on specific TP53 mutations involved [7,8]. The benefits of clinical surveillance of LFS patients with known pathogenic mutations in TP53 are well established [9,10], therefore it is important to identify patients with LFS as rapidly as possible to offer appropriate cancer surveillance and prophylaxis management as well as to extend genetic testing to at-risk family members.

Here we describe the function of a p53 variant, c.758C > T (T253I), observed in an infant who presented with ACC and a germline variant of uncertain significance (VUS) in TP53. While pediatric ACC is associated with LFS [11,12], and the p53 T253I mutation has been identified as a temperature sensitive mutation in yeast [13], there have been no additional published studies focusing on the functional impact of the T253I mutation on p53 in mammalian cells. By comparing the function of T253I in vitro to both p53 WT and two established loss of function p53 mutations also identified in pediatric patients (C176Y and R213X [14,15]), our findings suggest that T253I represents a pathogenic loss-of-function mutation in p53 that may be associated with LFS.

Materials and methods

Cells and cell culture

HEK TP53 wt/wt (ATCC Cat. #CRL-1573), HEK Crispr-deleted TP53-null (TP53^-/-; Ubigene Cat. #YKO-H177), and all stably transduced derivatives of HEK TP53^-/- cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FBS (Gemini Bio-Products) in a 37°C incubator with 5% CO₂ and maintained in log growth phase.

Stable expression mutant p53 genes

HEK p53^-/- cells were transduced with a lentivirus containing GFP-tagged p53 (p53 WT-GFP) or mutant derivatives of p53: C176Y-GFP, R213X-GFP or T253I-GFP, all under a cytomegalovirus (CMV) promoter (Origene Cat. # RC200003L4V). Stable lines were selected (puromycin) and expression of constructs confirmed by GFP fluorescence and immunoblotting.

Cell growth rate and GFP expression detection by flow cytometry

Doubling times for HEK p53^-/- and HEK p53^-/- cells complemented with p53 WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP were tested by CellTrace Violet proliferation assay (Thermo Fisher Cat. #C34571) as described [16].

Subcellular fractionation and immunoblotting

HEK p53^-/- and HEK p53^-/- cells complemented with p53 WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP were grown in log phase. Nuclear and cytoplasmic fractions were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific Cat. #78833) and immunoblotted for the indicated proteins using GAPDH and histone H3 as purity controls. For whole cell lysate experiments, immunoblotting was performed according to manufacturer’s instructions and published methods [17,18]. A full list of antibodies used is available in S1 Table.

RT-PCR analysis

Gene expression measurement for p21 and MDM2 was conducted using real-time polymerase chain reaction (rtPCR) analysis as previously described [18] under the following conditions: 25°C for 10 minutes, 42°C for 60 minutes, and 95°C for 5 minutes. Primer pairs and TaqMan probes (Thermo Scientific. Cat. # 4331182) were used to determine p21 (Cdkn1a Gene Expression Assay ID Hs00355782_ml) and MDM2 (Gene Expression Assay ID: Hs99999008_m1) gene expression levels. Gene expression data were normalized to 18s gene expression of eukaryotic 18S rRNA endogenous control (Thermo Scientific Cat. # 4319413E).

DNA binding assay

p53 DNA binding capacity was measured using a colorimetric p53 Transcription Factor ELISA Assay Kit (Abcam Cat. #ab207225) as previously published [19]. Signal strength was calculated for each p53 condition with or without damage, normalized using p53^-/- cells as a negative control and compared relative to undamaged WT-GFP-complemented p53^-/- cells (positive control).

Transcriptional activity assay

p53 transcriptional activity was measured using the Dual-Glo luciferase assay system (Promega Cat#E2940) as previously published [20] with two plasmids, luc2P/P53 response element (Promega Cat #E393A) and hRluc/CMV (Promega Cat. #E365A) using log-phase HEK p53^-/- and p53^-/- + WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP cells. Data reflect three experimental replicates, each with eight technical replicates for each cell line.

CHIP-PCR assay

The ability of p53 to bind in vitro to DNA was measured using chromatin immunoprecipitation and PCR amplification. Chromatin immunoprecipitation was performed using the SimpleChIP® Enzymatic Chromatin IP Kit (CST Cat #9003). Briefly, genomic DNA from whole cell extracts was crosslinked to all proteins actively binding DNA, fragmented into small (150–900 bp) lengths, then immunoprecipitated with p53 antibody or a positive control with H3 antibody (CST Cat #4620). After purification of the DNA fragments, PCR amplification using ChIP suitable p21 primers was performed p21 (FOR: CCC ACA GCA GAG GAG AAA GAA. REV: CTG GAA ATC TCT GCC CAG ACA. Sigma Aldrich Cat# 17–613), normalized to the input expression of RPL30 (Exon 3 Primers from Sigma Aldrich Cat# 7014S), for each p53 condition. Data reflect the mean and standard deviation of three experimental replicates.

Statistics

Statistical analyses were performed in conjunction with our institutional Biostatistics and Bioinformatics Shared Resource Facility. In all figures, data are presented as mean ± standard error of proportions (percents). One-Way ANOVA for multiple group comparisons was used to assess statistical significance of differences amongst and between groups of interest. An experiment-wise significance level of 5% was set prior to data analysis and appropriate post-hoc testing (Newman-Keuls Multiple Comparison Test) was employed. Where appropriate, statistical significance is denoted by symbols (* indicates p < 0.05 when comparing the sample to p53 -/-, † indicates p < 0.05 when comparing the sample to p53 WT). GraphPad Prism V 5.01 was used to perform the statistical analyses presented in this manuscript with the exception of the growth rates, which were determined by exponential decay regression analysis performed by Sigmaplot V 15.0. In this case, the amount of signal remaining at each timepoint was calculated as a percentage relative to the initial signal at time 0 and the half-life was found by solving the exponential decay regression curve y = a * exp(-b * x) for 50% signal remaining. This half-life value was then used to represent the population doubling time.

Ethical statement

This research was developed from a clinical study called “Project Inherited Cancer Risk” offered at the University of Kentucky to provide germline sequencing for a panel of cancer-associated genes. The study was vetted and approved by our Institutional Review Board (IRB), and permission for the use of the patients’ data for research purposes was explicitly granted by the consenting legal guardian.

Results

Detection of p53 T253I germline mutation in ACC patient

A pediatric patient was recently diagnosed with a stage 1 adrenocortical carcinoma. Tumor molecular profiling identified a c.758C > T variant in the TP53 gene (variant allele frequency 72%) resulting in a threonine to isoleucine substitution at position 253 of p53 protein. The resected ACC tumor tissue stained more strongly for total p53 protein levels than adjacent non-neoplastic tissue, and the staining was strongly nuclear (S1 Fig), similar to tumor histologies of p53-mutant tumors in previously published studies [21]. ACC is a tumor characteristic of LFS, a cancer predisposition syndrome caused by germline mutations that disrupt p53 tumor suppressor function [1,2]. Indeed, 27 of 39 pediatric ACC patients had mutations in TP53, an incidence rate of almost 70% in a hallmark study of pediatric cancers [12]. The TP53 c.758C > T variant was also found in the patient’s germline, however since we observed that the TP53 c.758C > T T253I variant is classified as a variant of uncertain significance (VUS) in the ClinVar database, we therefore sought to characterize the impact of the T253I mutation on p53 function.

Stable expression of clinically observed p53 mutants

We hypothesized that the p53 T253I mutation may result in a loss of function in the tumor suppressor p53 since pediatric ACC can be associated with LFS [22,23]. To test this, we developed TP53 CRISPR-deleted HEK293 cells (HEK p53^-/-) as a system in which p53-null cells could be complemented with wild type (WT) or mutant p53 to compare cell growth and p53-mediated damage responses. We observed no p53 expression in HEK p53^-/- cells in contrast to unmanipulated HEK293 p53 ^+/+ cells or WT-complemented p53^-/- HEK293 cells (S2 Fig). We found that the GFP-tagged p53 WT band was of higher molecular weight, about 75 kDa compared to 53 kDa, than native p53 WT, likely because of the GFP tag attached to p53 constructs in the complemented condition. Further, we noted that expression of WT-GFP p53 was considerably stronger than native p53 present in parental TP53-intact HEK293 cells (6.1-fold, comparing lanes 1 and 3, p < 0.05), presumably because expression of the construct was under the control of a CMV promotor.

We next developed a T253I TP53 construct as well as two known loss-of-function p53 mutants for use as experimental controls – C176Y [24,25] and R213X [26,27] (S3 Fig). Each GFP-tagged TP53 construct was independently and stably transduced into TP53-null HEK293 cells by lentiviral-mediated delivery. We took advantage of the GFP-tags on each of the complemented constructs to verify expression and to measure p53 construct expression through cellular green fluorescence by flow cytometry (Fig 1). Uncomplemented p53^-/- cells had negligible levels of fluorescence at 488 nm, while HEK293 cells complemented with WT-GFP, C176Y-GFP, R213X-GFP and T253I-GFP p53 constructs each exhibited stronger expression of green fluorescence (Fig 1a). We observed that the expression of each of the p53 mutants tested (C176Y-GFP, R213X-GFP, T253I-GFP) was significantly higher than that of the wild type p53-GFP complemented condition, with median fold induction values of 3.0, 2.6 and 4.1 (p < 0.05) compared to p53 T253-GFP (WT), respectively (Fig 1b). To confirm that these levels of p53 were not detrimental to normal cellular growth, we performed a dye dilution assay to measure cell doubling times using the Cell Trace Violet Proliferation Kit (Thermo Scientific). Population doubling time was not statistically different (p = .7903, 1-Way ANOVA) between HEK p53^-/- cells and p53^-/- cells complemented with either p53 WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP (Fig 1c). Therefore, we concluded that expression of any of the p53 constructs did not impact cell proliferation.

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Fig 1. Stable incorporation of p53 lentiplasmids is detectible and does not impact cellular growth rates.

(A) HEK293 p53^-/- cells were stably complemented either WT-GFP, C176Y-GFP, R213X-GFP or T253I-GFP p53. After selection, cells were assayed for GFP fluorescence by flow cytometry. Shown are representative histograms (green fluorescence) of the geometric means of each complemented cell type. (B) Graphical representation of fluorescent intensity of each p53 cell line. Median GFP fluorescent intensity was calculated for each cell line and normalized to the p53 WT expression levels. Data represent the mean of three experimental replicates. (C) Cell growth kinetics of stably complemented HEK293 p53^-/- cells were measured by use of a CellTrace Violet assay. After incorporating a fluorescent dye, cells were harvested 24, 48, 72, and 96 hours after treatment, fixed, and dye intensity was measured by flow cytometry. Geometric mean intensity at each timepoint was used to calculate an exponential decay regression curve and ultimately a population doubling time (PDT). Data represent the mean PDT of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

https://doi.org/10.1371/journal.pone.0320036.g001

T253I shows abnormal autoregulatory feedback with MDM2

Given the higher levels of GFP signal in the mutant p53 expressing cells, we measured total p53 protein levels by immunoblotting in p53^-/- HEK293 cells and in those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53. Whereas no p53 protein was found in in p53^-/- HEK293 cells, there was evidence of p53 expression in each of the complemented cell lines. Similar to findings with GFP fluorescence by flow cytometry, we observed that levels of expression of C176Y-GFP, R213X-GFP, or T253I-GFP p53 were higher than that of WT-GFP with densitometry values 4.5-, 1.8- and 3.2-fold higher than p53 WT-GFP p53, respectively (S4 Fig).

We then considered why protein levels of p53 T253I-GFP, like those of C176Y-GFP and R213X-GFP, were stronger than p53 WT-GFP levels despite each being expressed under the same (CMV) promoter. Knowing that p53 levels are regulated mainly by an auto-feedback mechanism wherein p53 transcriptionally induces expression of MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation [28,29], we surmised that overexpression of mutant p53 proteins may be due to lower MDM2 levels, presumably because of loss of p53 transcriptional activity. Indeed, we observed that MDM2 levels were substantially lower in the p53^-/- null (12.5%) and C176Y-GFP, R213X-GFP, or T253I-GFP p53 cells (15.3%, 19.5%, and 34% for p53 C176Y-GFP, R213X-GFP, and T253I-GFP, respectfully compared to p53 WT-GFP complemented cells; S4 Fig). We interpret these data to indicate that the high p53 expression levels of the mutant p53 proteins may be a consequence of insufficient MDM2 levels in the setting of defective p53 transcriptional activity, interfering with the autoregulatory feedback loop’s regulation of cellular p53 levels.

Since p53 nuclear localization is critical to its function and is one of the targets for MDM2 negative regulation, we next investigated the cytoplasmic and nuclear distribution of p53 and MDM2 in p53^-/- null, WT-GFP, or C176Y-GFP, R213X-GFP, or T253I-GFP complemented HEK293 cells. We observed that each of the p53 constructs could be detected in both nuclear and cytoplasmic fractions (Fig 2a) with cytoplasmic levels (Fig 2b, left) of each of the mutants higher than that of WT (fold increases of 2.6, 2.2 and 2.7 respectively). In contrast, nuclear levels (Fig 2b, right) of each of the mutants were much more similar to that of WT p53 (fold changes of 1.4, 1.0, and 1.5 respectively). With respect to MDM2 localization, cytoplasmic MDM2 levels were similar across all conditions examined (Fig 2c, left), while nuclear MDM2 levels were higher in the WT-complemented condition (Fig 2c, right). Indeed, each mutant line had nuclear MDM2 levels comparable to uncomplemented p53^-/- cells, suggesting p53-mediated dysfunctional MDM2 localization in the setting of any of the mutants tested (C176Y, R213X, or T253I). We conclude that reduced nuclear MDM2 levels in p53 T253I-GFP complemented cells, similar to the p53-null or loss-of-function p53 control conditions (C176Y-GFP, R213X-GFP), may contribute to the increased total p53 expression levels by an impaired p53-MDM2 autoregulatory loop. Further, these findings suggest that the T253I mutation may impede p53 transactivation ability and MDM2 nuclear localization.

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Fig 2. Subcellular fractionation of HEK293 p53 wt and mutant proteins.

HEK293 p53^-/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were subjected to subcellular fractionation to look at the distribution of p53 and MDM2 in the cytoplasm (A, left side) and nucleus (A, right side). (B) Graphical depictions of the cytoplasmic (left) and nuclear (right) expression levels of p53. (C) Graphical depictions of the cytoplasmic (left) and nuclear (right) expression levels of MDM2. In both cases, expression levels of cytoplasmic fractions were normalized to GAPDH while expression levels of nuclear fractions were normalized to Histone 3 (H3). Data represent the mean expression levels of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

https://doi.org/10.1371/journal.pone.0320036.g002

Canonical p53 DNA damage responses are negatively impacted in p53 T253I cells

To test the impact of the T253I mutation on p53 protein function, we measured p53-dependent and damage-mediated induction of MDM2 and p21 [30] by incubating HEK293 p53^-/- cells complemented with WT-GFP- or C176Y-GFP, R213X-GFP or T253I-GFP p53 in the presence or absence of sublethal cisplatin. Kinetic studies in p53 WT-GFP cells documented significant accumulation of p21 and MDM2 protein by 2h after cisplatin exposure (S5 Fig), therefore we chose the 2h time point to characterize the impact of each of the mutations on p53 function. We observed that levels of total p53 were higher across all mutations tested compared to WT and that cisplatin did not affect total p53 levels (Fig 3a, 3b). Damage-induced up-regulation of MDM2 and p21 signals was observed in WT-GFP-complemented p53^-/- cells, but not in p53^-/- or C176Y-GFP, R213X-GFP or T253I-GFP-complemented p53^-/- cells HEK293 cells (Fig 3a, 3c, 3d). Additionally, MDM2 and p21 levels in undamaged WT-GFP complemented p53^-/- cells were significantly stronger than in the p53 mutant or null cell lines. Together these observations suggest that the T253I mutation, like known loss-of-function C176Y and R213X mutations, interferes with canonical p53 damage responses.

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Fig 3. Impact of p53 T253I on expression of DNA damage responsive proteins.

HEK293 p53^-/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were treated with 5 ug/mL Cisplatin (or a DMSO vehicle control) for one hour followed by a 2-hour recovery period. Cells were collected and a Western blot was run looking at p53 signaling events. Levels of p53, MDM2, (A, left side) and p21 (A, right side) were examined with or without damage in each of the p53 conditions (null, WT, and the three mutants). Graphical depictions of p53 (B), p21 (C), and MDM2 (D) are shown. Data represent the mean expression levels, normalized to GAPDH, of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

https://doi.org/10.1371/journal.pone.0320036.g003

Given that p53 transcriptionally regulates p21 and MDM2 expression [30], we compared the ability of the T253I mutant to WT, C176Y and R213X p53 to modulate expression of p21 and MDM2 mRNA by real time PCR. We observed higher baseline of p21 (p < 0.05) and MDM2 (higher, but not significant) mRNA expression in WT-GFP-complemented p53^-/- cells as compared to C176Y-GFP, R213X-GFP or T253I-GFP-complemented p53^-/- cells HEK293 cells (Fig 4a, 4b). With damage, both p21 and MDM2 mRNA expression levels increase, but not significantly. These data suggest that the T253I mutation, like C176Y and R213X known loss-of-function variants, impairs p53 transcriptional activity.

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Fig 4. Impact of p53 T253I on DNA damage responsive mRNA expression.

HEK293 p53^-/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were treated with 5 ug/mL Cisplatin (or a DMSO vehicle control) for one hour followed by a 2-hour recovery period. RNA was isolated from these cells and subjected to rtPCR in the presence of primers for p21 (A) and MDM2 (B). Data represent the mean RNA expression levels of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

https://doi.org/10.1371/journal.pone.0320036.g004

Next, we compared the ability of the p53 constructs to bind a consensus p53 DNA binding sequence by ELISA. P53 WT-GFP binds to this DNA much more strongly than any of the other mutants tested (58.69%, 49.90%, and 61.43% lower for C176Y-GFP, R213X-GFP or T253I-GFP respectively; Fig 5a). In addition, sublethal cisplatin exposure increased binding of WT p53 by 92.6% (Fig 5a, columns 1 and 2) but did not increase binding of any of the mutants tested, suggesting T253I has impaired basal- and DNA damage-induced DNA binding capabilities. We also performed a CHIP-seq assay to measure the ability of p53 constructs to bind genomic DNA at the p21 promoter (Fig 5b). Whereas HEK p53 ^-/-- cells possessed no binding capabilities, those complemented with p53-WT showed strong binding signals. In contrast, all p53 mutant cell lines (C176Y, R213X, and T253I) had significantly reduced p53 binding capability (68–80% reduction in DNA binding compared to WT). In order to directly compare transcriptional regulatory capacity of T253I with WT p53, we employed a dual luciferase reporter assay consisting of a p53-dependent promotor driving Firefly luciferase to measure p53-dependent responses, and a CMV promotor-driven Renilla luciferase to control for transfection. Whereas strong p53-dependent firefly luciferase signal was observed from p53-null HEK293 cells complemented with WT-GFP, there was minimal signal from either uncomplemented p53^-/- cells or from p53^-/- cells complemented with either C176Y-GFP, R213X-GFP or T253I-GFP (S6 Fig). Since all cell conditions had strong signal from the CMV-promoter driven Renilla luciferase, we concluded that observed differences were not caused by differential transfection efficiency in the assay. These data indicate that WT-complemented p53^-/- cells alone had transcriptionally active p53 protein in this assay (Fig 5c). We conclude that although the T253I mutant p53 protein (like the C176Y or R213X mutant p53 proteins) may bind DNA in a limited capacity, its transcriptional activity is markedly impaired.

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Fig 5. Mutant p53 proteins have impaired DNA binding and transcriptional regulation.

HEK293 p53^-/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were assayed for DNA binding capability and transcriptional regulatory capacity. (A) Log phase cells were exposed 5 µg/mL Cisplatin (or a DMSO vehicle control) for 24 hours. Nuclear protein was isolated from cells and a DNA binding assay was performed. Binding efficiency was measured by the ability of nuclear p53 to recognize and bind to consensus p53 binding DNA sequences attached to 96-well plate wells. Data represent the binding strength of each condition relative to undamaged p53 WT cells and are expressed as the mean of three experimental replicates. (B) DNA binding capacity of HEK p53^-/- and p53 ^-/- + WT, C176Y, R231X, or T253I p53 log phase cells was measured using CHIP-PCR. Genomic DNA was isolated, fragmented, and immunprecipitated with p53 antibody. After separating the DNA, primers to p21 (and RPL30 as a control) were used to identify the relative quantity of bound p21 promoter DNA in each p53 condition through PCR. Data represent the mean of three experimental replicates. (C) Transcriptional regulatory capacity was measured using a dual-glow luciferase assay. Cells were co-transfected with luc2P/p53 RE and hRluc/CMV vectors at a 10:1 ratio. After 24 hours the expression of these genes was measured by a stop-and-glow (Promega) system. For each p53 condition, luminescent intensity was calculated as a ratio of luc2P to hRluc and expressed as a percentage of intensity relative to WT-GFP-complemented p53^-/- cells. Data are expressed as the mean of three experimental replicates and significance in (A) (B) and (C) is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

https://doi.org/10.1371/journal.pone.0320036.g005

Discussion

Here we report evidence supporting the designation of p53 T253I as a LFS-associated loss of function mutation. Observed in a young child with an ACC and germline c.758C > T TP53 variant, the resulting T253I mutant p53 protein exhibited attenuated p53 baseline and damage-induced cell responses. The strong clinical association between pediatric ACC and Li-Fraumeni Syndrome (LFS) [31], coupled with the paradoxical overexpression of p53 in the tumor, led us to hypothesize that the p53 T253I mutation may be pathogenic and represent a loss-of-function mutation, similar to R213X and C176Y, two other known LFS-associated variants [14,15]. LFS is a cancer predisposition syndrome associated with life-long higher risk of malignancy [3,4], and patient outcomes have been shown to be improved by appropriate cancer surveillance [9,10,32]. Our goal was to establish T253I as an LFS-associated TP53 mutation to justify personalized cancer surveillance and appropriate genetic counseling for our patient and other patients with the same germline variant.

Loss-of-function mutations in TP53 may contribute to carcinogenesis through interference with p53-mediated DNA damage responses [33]. Consistent with our observations of T253I both in the cancer tissue and cultured cells, many loss-of-function p53 mutations are known to result in high cellular levels of the mutant p53 protein because of impaired p53-induced MDM2 induction and reduced autoregulatory feedback [34]. Although it has been suggested that elevated mutant p53 levels may function in an oncogenic manner by exerting antimorphic (dominant negative) effects on residual WT p53 [35–39], recent reports have argued against this possibility [40], and our studies have not clarified whether T253I p53 may impact residual WT p53 function. We realize that the cell responses of mutant TP53 in the background of an otherwise TP53-deficient cell (as in our studies) may differ from heterozygous expression as would be expected in germline LFS carriers. However, we reasoned that the p53 null background would enable clarity of the impact of p53 T253I on p53 tumor suppressor function. We acknowledge that our study design did not investigate the impact of each variant studied on cellular damage and survival responses in the heterozygous setting which would model the germline LFS state. Instead, we decided to interrogate the T253I variant to understand its impact on p53 responses in isolation, but we are planning follow-up studies to determine how T253I p53 interacts with wt p53 and its impact on cell responses in a heterozygous setting.

Using the system of p53 complementation into a p53 ^-/- background, we found evidence that p53 T253I exhibits attenuated p53 signaling responses including damage-induced p21 and MDM2 induction and reduced DNA binding and transcriptional activity compared to p53 WT, comparable to established LOF mutants (Figs 3–5). We realize that tagging p53 proteins with GFP may alter their behavior, however we observed that GFP-tagged p53 WT cells complemented the loss of p53 signaling seen in p53 -/- cells, and that GFP-tagged p53 mutant cells exhibit attenuated responses. Since each p53 mutant contained the identical GFP tag as the wt p53, we consider the presence of a GFP tag to be unlikely to account for attenuated responses of any of the p53 mutants and conclude that this issue does not detract from the primary observation in the manuscript (i.e., that T253I p53 exhibits loss of function).

Together with the clinical observation of an LFS-characteristic tumor in a patient with a germline p53 T253I mutation, our studies suggest that p53 T253I results in a loss of p53 function and may establish a pro-carcinogenic cellular state consistent with a cancer predisposition syndrome such as LFS. It is important to point out that while the patient harboring this mutation developed an ACC at a very young age, the patient’s father – the only other member of the family confirmed as a carrier of TP53 c.758C > T (T253I) – has not presented with cancer to date. This raises the possibility that that germline p53 T253I may be variably penetrant, consistent with prior observations of hypomorphic p53 mutations in ACCs [41], or it could simply be that that father has not developed a cancer yet and remains at high risk of doing so.

As more genetic variants are identified in cancer-relevant genes, it is increasingly important to determine functional significance to clinically observed mutations to guide clinical management as well as to gain insight into disease pathophysiology. Proper classification of the p53 T253I mutation as an LFS-associated pathogenic variant is important to develop a rational cancer-preventive and early surveillance plan to improve clinical outcomes for our patient, their family and for other patients with germline p53 T253I mutations.

Supporting information

S1 Table. An itemized list of antibodies used in this manuscript.

https://doi.org/10.1371/journal.pone.0320036.s001

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S1 Fig. Histological staining of tumor tissue section shows classical signs of ACC as well as suggests p53 abnormalities.

(Top) Immunohistochemical staining of the ACC showing that tumor cells were immunoreactive for synaptophysin and Melan-A, with increased Ki67; total tissue Ki67 positivity was estimated at 15%. (Bottom) Neoplastic tissue contained higher levels of total p53 protein than adjacent non-neoplastic tissue.

https://doi.org/10.1371/journal.pone.0320036.s002

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S2 Fig. Complementation of HEK p53^-/- cells with p53 WT-GFP.

(A) A Western blot of HEK p53 ^+/+, HEK293 p53^-/-, and WT-GFP-complemented HEK p53^-/- cells, as well as a graphical depiction (B) of the relative abundances of p53 in each cell line. Data represent the mean expression levels, normalized to GAPDH, of at least three experimental replicates.

https://doi.org/10.1371/journal.pone.0320036.s003

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S3 Fig. Location of incorporation of p53 mutants into HEK293 cells.

A cartoon schematic of the functional domains of the p53 protein overlaid with the locations of the mutations being examined in this study. Note that all three mutations lie within the DNA binding domain and that both single nucleotide polymorphisms (C176Y and T253I) are further located within highly conserved regions of the DNA binding domain of p53.

https://doi.org/10.1371/journal.pone.0320036.s004

(PDF)

S4 Fig. Steady state expression levels of p53 and MDMD2 in HEK293 p53^-/- cells complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53.

A Western blot of HEK p53^-/- with or without complementation with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 as well as a graphical depiction of the relative abundances (B) of p53 and MDM2 in each cell line. Data represent the mean expression levels, normalized to GAPDH, of three experimental replicates.

https://doi.org/10.1371/journal.pone.0320036.s005

(PDF)

S5 Fig. Timecourse of recovery after cisplatin treatment shows maximal induction window for p53-dependent DNA damage responses.

(A) WT-GFP-complemented HEK p53^-/- cells were treated with 5 µg/mL for one hour (or untreated) and then allowed to recover for a period of time ranging from 1–24 hours. At the end of the recovery period, cells were harvested and a Western Blot was run to measure the expression of p53, MDM2, and p21, using GAPDH as a loading control. (B) A graphical depiction of the relative abundances of p53, MDM2, and p21, normalized to GAPDH, at each timepoint after damage for the duration of the recovery period. Data were presented as the percentage of expression relative to non-damaged (ND) cells and represent the average of three replicates.

https://doi.org/10.1371/journal.pone.0320036.s006

(PDF)

S6 Fig. Luciferase signal strength from luc2P/p53 RE and hRluc/CMV reporters.

(A) p53-driven firefly luciferase reporter assay and (B) CMV-driven renilla reporter assay results that were combined to generate the ratio as seen in Fig 5b. Data represent the mean luciferase signal, normalized to p53 WT-GFP expressing cells, of three experimental replicates. Samples marked with an * are not significantly different from each other.

https://doi.org/10.1371/journal.pone.0320036.s007

(PDF)

Acknowledgments

We are grateful to the Biostatistics and Bioinformatics, Flow Cytometry and Immune Monitoring, Biospecimen Procurement and Translational Pathology, Cancer Research and Informatics, and Oncogenomics Shared Resource Facilities of the University of Kentucky Markey Cancer Center for their technical and scientific contributions to the work. We thank the COBRE Imaging Core facility at the University of Kentucky for providing service with microscopes and imaging systems. We are indebted to the Clinical Research Office of the Division of Pediatric Hematology/Oncology at the University of Kentucky for their efforts in accrual and data management for Project Inherited Cancer Risk, the clinical study to identify and manage children, adolescents and young adults with cancer predisposition syndromes. Finally, we are immensely grateful to the patients and families who consented to take part in the “Project Inherited Cancer Risk” study at the University of Kentucky by which these variants were identified.

References

1. Li FP, Fraumeni JF Jr. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med. 1969;71(4):747–52. pmid:5360287
- View Article
- PubMed/NCBI
- Google Scholar
2. Schneider K, Zelley K, Nichols KE, Garber J. Li-Fraumeni Syndrome. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, editors. GeneReviews(R). Seattle (WA): 1993.
3. Ballinger ML, Mitchell G, Thomas DM. Surveillance recommendations for patients with germline TP53 mutations. Curr Opin Oncol. 2015;27(4):332–7. pmid:26049273
- View Article
- PubMed/NCBI
- Google Scholar
4. Correa H. Li-Fraumeni Syndrome. J Pediatr Genet. 2016;5(2):84–8. pmid:27617148
- View Article
- PubMed/NCBI
- Google Scholar
5. Nichols KE, Malkin D, Garber JE, Fraumeni JF Jr, Li FP. Germ-line p53 mutations predispose to a wide spectrum of early-onset cancers. Cancer Epidemiol Biomarkers Prev. 2001;10(2):83–7. pmid:11219776
- View Article
- PubMed/NCBI
- Google Scholar
6. Shin SJ, Dodd-Eaton EB, Peng G, Bojadzieva J, Chen J, Amos CI, et al. Penetrance of different cancer types in families with Li-Fraumeni Syndrome: a validation study using multicenter cohorts. Cancer Res. 2020;80(2):354–60. pmid:31719101
- View Article
- PubMed/NCBI
- Google Scholar
7. Rocca V, Blandino G, D’Antona L, Iuliano R, Di Agostino S. Li-Fraumeni Syndrome: mutation of TP53 is a biomarker of hereditary predisposition to tumor: new insights and advances in the treatment. Cancers (Basel). 2022;14(15):3664. pmid:35954327
- View Article
- PubMed/NCBI
- Google Scholar
8. Gargallo P, Yáñez Y, Segura V, Juan A, Torres B, Balaguer J, et al. Li-Fraumeni syndrome heterogeneity. Clin Transl Oncol. 2020;22(7):978–88. pmid:31691207
- View Article
- PubMed/NCBI
- Google Scholar
9. Villani A, Tabori U, Schiffman J, Shlien A, Beyene J, Druker H, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: a prospective observational study. Lancet Oncol. 2011;12(6):559–67. pmid:21601526
- View Article
- PubMed/NCBI
- Google Scholar
10. Villani A, Shore A, Wasserman JD, Stephens D, Kim RH, Druker H, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: 11 year follow-up of a prospective observational study. Lancet Oncol. 2016;17(9):1295–305. pmid:27501770
- View Article
- PubMed/NCBI
- Google Scholar
11. Gröbner SN, Worst BC, Weischenfeldt J, Buchhalter I, Kleinheinz K, Rudneva VA, et al. The landscape of genomic alterations across childhood cancers. Nature. 2018;555(7696):321–7. pmid:29489754
- View Article
- PubMed/NCBI
- Google Scholar
12. Zhang J, Walsh MF, Wu G, Edmonson MN, Gruber TA, Easton J, et al. Germline mutations in predisposition genes in pediatric cancer. N Engl J Med. 2015;373(24):2336–46. pmid:26580448
- View Article
- PubMed/NCBI
- Google Scholar
13. Shiraishi K, Kato S, Han S-Y, Liu W, Otsuka K, Sakayori M, et al. Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library. J Biol Chem. 2004;279(1):348–55. pmid:14559903
- View Article
- PubMed/NCBI
- Google Scholar
14. Zhang Y, Zhang Y, Zhao H, Zhai Q, Zhang Y, Shen Y. The impact of R213 mutation on p53-mediated p21 activity. Biochimie. 2014;99:215–8. pmid:24384472
- View Article
- PubMed/NCBI
- Google Scholar
15. Hassan NMM, Tada M, Hamada J, Kashiwazaki H, Kameyama T, Akhter R, et al. Presence of dominant negative mutation of TP53 is a risk of early recurrence in oral cancer. Cancer Lett. 2008;270(1):108–19. pmid:18555592
- View Article
- PubMed/NCBI
- Google Scholar
16. Filby A, Begum J, Jalal M, Day W. Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution. Methods. 2015;82:29–37. pmid:25802116
- View Article
- PubMed/NCBI
- Google Scholar
17. Choi HY, Lim JE, Hong JH. Curcumin interrupts the interaction between the androgen receptor and Wnt/β-catenin signaling pathway in LNCaP prostate cancer cells. Prostate Cancer Prostatic Dis. 2010;13(4):343–9. pmid:20680030
- View Article
- PubMed/NCBI
- Google Scholar
18. Pu H, Horbinski C, Hensley PJ, Matuszak EA, Atkinson T, Kyprianou N. PARP-1 regulates epithelial-mesenchymal transition (EMT) in prostate tumorigenesis. Carcinogenesis. 2014;35(11):2592–601. pmid:25173886
- View Article
- PubMed/NCBI
- Google Scholar
19. Kang Y-J, Yang W-G, Chae W-S, Kim D-W, Kim S-G, Rotaru H. Administration of 4‑hexylresorcinol increases p53‑mediated transcriptional activity in oral cancer cells with the p53 mutation. Oncol Rep. 2022;48(3):160. pmid:35856441
- View Article
- PubMed/NCBI
- Google Scholar
20. Yuan M, Liu X, Wang M, Li Z, Li H, Leng L, et al. A functional variant alters the binding of bone morphogenetic protein 2 to the Transcription Factor NF-κB to regulate bone morphogenetic protein 2 gene expression and chicken abdominal fat deposition. Animals (Basel). 2023;13(21):3401. pmid:37958155
- View Article
- PubMed/NCBI
- Google Scholar
21. Ribeiro RC, Sandrini F, Figueiredo B, Zambetti GP, Michalkiewicz E, Lafferty AR, et al. An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma. Proc Natl Acad Sci U S A. 2001;98(16):9330–5. pmid:11481490
- View Article
- PubMed/NCBI
- Google Scholar
22. Wasserman JD, Novokmet A, Eichler-Jonsson C, Ribeiro RC, Rodriguez-Galindo C, Zambetti GP, et al. Prevalence and functional consequence of TP53 mutations in pediatric adrenocortical carcinoma: a children’s oncology group study. J Clin Oncol. 2015;33(6):602–9. pmid:25584008
- View Article
- PubMed/NCBI
- Google Scholar
23. Varley JM. Germline TP53 mutations and Li-Fraumeni syndrome. Hum Mutat. 2003;21(3):313–20. pmid:12619118
- View Article
- PubMed/NCBI
- Google Scholar
24. Kotler E, Shani O, Goldfeld G, Lotan-Pompan M, Tarcic O, Gershoni A, et al. A systematic p53 mutation library links differential functional impact to cancer mutation pattern and evolutionary conservation. Mol Cell. 2018;71(5):873. pmid:30193102
- View Article
- PubMed/NCBI
- Google Scholar
25. Mullany LK, Wong K-K, Marciano DC, Katsonis P, King-Crane ER, Ren YA, et al. Specific TP53 mutants overrepresented in ovarian cancer impact CNV, TP53 activity, responses to nutlin-3a, and cell survival. Neoplasia. 2015;17(10):789–803. pmid:26585234
- View Article
- PubMed/NCBI
- Google Scholar
26. Masciari S, Dewanwala A, Stoffel EM, Lauwers GY, Zheng H, Achatz MI, et al. Gastric cancer in individuals with Li-Fraumeni syndrome. Genet Med. 2011;13(7):651–7. pmid:21552135
- View Article
- PubMed/NCBI
- Google Scholar
27. Ruijs MWG, Verhoef S, Rookus MA, Pruntel R, van der Hout AH, Hogervorst FBL, et al. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes. J Med Genet. 2010;47(6):421–8. pmid:20522432
- View Article
- PubMed/NCBI
- Google Scholar
28. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2. Nature. 1997;387(6630):299–303. pmid:9153396
- View Article
- PubMed/NCBI
- Google Scholar
29. Alarcon-Vargas D, Ronai Z. p53-Mdm2--the affair that never ends. Carcinogenesis. 2002;23(4):541–7. pmid:11960904
- View Article
- PubMed/NCBI
- Google Scholar
30. Chen J. The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harb Perspect Med. 2016;6(3):a026104. pmid:26931810
- View Article
- PubMed/NCBI
- Google Scholar
31. Wagner J, Portwine C, Rabin K, Leclerc JM, Narod SA, Malkin D. High frequency of germline p53 mutations in childhood adrenocortical cancer. J Natl Cancer Inst. 1994;86(22):1707–10. pmid:7966399
- View Article
- PubMed/NCBI
- Google Scholar
32. Kratz CP, Villani A, Nichols KE, Schiffman J, Malkin D. Cancer surveillance for individuals with Li-Fraumeni syndrome. Eur J Hum Genet. 2020;28(11):1481–2. pmid:32814850
- View Article
- PubMed/NCBI
- Google Scholar
33. Freed-Pastor WA, Prives C. Mutant p53: one name, many proteins. Genes Dev. 2012;26(12):1268–86. pmid:22713868
- View Article
- PubMed/NCBI
- Google Scholar
34. Prives C, White E. Does control of mutant p53 by Mdm2 complicate cancer therapy? Genes Dev. 2008;22(10):1259–64. pmid:18483214
- View Article
- PubMed/NCBI
- Google Scholar
35. Willis A, Jung EJ, Wakefield T, Chen X. Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes. Oncogene. 2004;23(13):2330–8. pmid:14743206
- View Article
- PubMed/NCBI
- Google Scholar
36. Chen X, Zhang T, Su W, Dou Z, Zhao D, Jin X, et al. Mutant p53 in cancer: from molecular mechanism to therapeutic modulation. Cell Death Dis. 2022;13(11):974. pmid:36400749
- View Article
- PubMed/NCBI
- Google Scholar
37. Zhang C, Liu J, Xu D, Zhang T, Hu W, Feng Z. Gain-of-function mutant p53 in cancer progression and therapy. J Mol Cell Biol. 2020;12(9):674–87. pmid:32722796
- View Article
- PubMed/NCBI
- Google Scholar
38. Muller PAJ, Vousden KH. p53 mutations in cancer. Nat Cell Biol. 2013;15(1):2–8. pmid:23263379
- View Article
- PubMed/NCBI
- Google Scholar
39. Inoue K, Kurabayashi A, Shuin T, Ohtsuki Y, Furihata M. Overexpression of p53 protein in human tumors. Med Mol Morphol. 2012;45(3):115–23. pmid:23001293
- View Article
- PubMed/NCBI
- Google Scholar
40. Wang Z, Burigotto M, Ghetti S, Vaillant F, Tan T, Capaldo BD, et al. Loss-of-function but not gain-of-function properties of mutant TP53 are critical for the proliferation, survival, and metastasis of a broad range of cancer cells. Cancer Discov. 2024;14(2):362–79. pmid:37877779
- View Article
- PubMed/NCBI
- Google Scholar
41. Jeffers JR, Pinto EM, Rehg JE, Clay MR, Wang J, Neale G, et al. The common germline TP53-R337H mutation is hypomorphic and confers incomplete penetrance and late tumor onset in a mouse model. Cancer Res. 2021;81(9):2442–56. pmid:33637564
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Li FP, Fraumeni JF Jr. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Intern Med. 1969;71(4):747–52. pmid:5360287
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Schneider K, Zelley K, Nichols KE, Garber J. Li-Fraumeni Syndrome. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, editors. GeneReviews(R). Seattle (WA): 1993.

[ref3] 3. Ballinger ML, Mitchell G, Thomas DM. Surveillance recommendations for patients with germline TP53 mutations. Curr Opin Oncol. 2015;27(4):332–7. pmid:26049273
View Article
PubMed/NCBI
Google Scholar

[7] View Article

[8] PubMed/NCBI

[9] Google Scholar

[ref4] 4. Correa H. Li-Fraumeni Syndrome. J Pediatr Genet. 2016;5(2):84–8. pmid:27617148
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Nichols KE, Malkin D, Garber JE, Fraumeni JF Jr, Li FP. Germ-line p53 mutations predispose to a wide spectrum of early-onset cancers. Cancer Epidemiol Biomarkers Prev. 2001;10(2):83–7. pmid:11219776
View Article
PubMed/NCBI
Google Scholar

[15] View Article

[16] PubMed/NCBI

[17] Google Scholar

[ref6] 6. Shin SJ, Dodd-Eaton EB, Peng G, Bojadzieva J, Chen J, Amos CI, et al. Penetrance of different cancer types in families with Li-Fraumeni Syndrome: a validation study using multicenter cohorts. Cancer Res. 2020;80(2):354–60. pmid:31719101
View Article
PubMed/NCBI
Google Scholar

[19] View Article

[20] PubMed/NCBI

[21] Google Scholar

[ref7] 7. Rocca V, Blandino G, D’Antona L, Iuliano R, Di Agostino S. Li-Fraumeni Syndrome: mutation of TP53 is a biomarker of hereditary predisposition to tumor: new insights and advances in the treatment. Cancers (Basel). 2022;14(15):3664. pmid:35954327
View Article
PubMed/NCBI
Google Scholar

[23] View Article

[24] PubMed/NCBI

[25] Google Scholar

[ref8] 8. Gargallo P, Yáñez Y, Segura V, Juan A, Torres B, Balaguer J, et al. Li-Fraumeni syndrome heterogeneity. Clin Transl Oncol. 2020;22(7):978–88. pmid:31691207
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 9. Villani A, Tabori U, Schiffman J, Shlien A, Beyene J, Druker H, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: a prospective observational study. Lancet Oncol. 2011;12(6):559–67. pmid:21601526
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Villani A, Shore A, Wasserman JD, Stephens D, Kim RH, Druker H, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: 11 year follow-up of a prospective observational study. Lancet Oncol. 2016;17(9):1295–305. pmid:27501770
View Article
PubMed/NCBI
Google Scholar

[35] View Article

[36] PubMed/NCBI

[37] Google Scholar

[ref11] 11. Gröbner SN, Worst BC, Weischenfeldt J, Buchhalter I, Kleinheinz K, Rudneva VA, et al. The landscape of genomic alterations across childhood cancers. Nature. 2018;555(7696):321–7. pmid:29489754
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 12. Zhang J, Walsh MF, Wu G, Edmonson MN, Gruber TA, Easton J, et al. Germline mutations in predisposition genes in pediatric cancer. N Engl J Med. 2015;373(24):2336–46. pmid:26580448
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Shiraishi K, Kato S, Han S-Y, Liu W, Otsuka K, Sakayori M, et al. Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library. J Biol Chem. 2004;279(1):348–55. pmid:14559903
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Zhang Y, Zhang Y, Zhao H, Zhai Q, Zhang Y, Shen Y. The impact of R213 mutation on p53-mediated p21 activity. Biochimie. 2014;99:215–8. pmid:24384472
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Hassan NMM, Tada M, Hamada J, Kashiwazaki H, Kameyama T, Akhter R, et al. Presence of dominant negative mutation of TP53 is a risk of early recurrence in oral cancer. Cancer Lett. 2008;270(1):108–19. pmid:18555592
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Filby A, Begum J, Jalal M, Day W. Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution. Methods. 2015;82:29–37. pmid:25802116
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Choi HY, Lim JE, Hong JH. Curcumin interrupts the interaction between the androgen receptor and Wnt/β-catenin signaling pathway in LNCaP prostate cancer cells. Prostate Cancer Prostatic Dis. 2010;13(4):343–9. pmid:20680030
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Pu H, Horbinski C, Hensley PJ, Matuszak EA, Atkinson T, Kyprianou N. PARP-1 regulates epithelial-mesenchymal transition (EMT) in prostate tumorigenesis. Carcinogenesis. 2014;35(11):2592–601. pmid:25173886
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref19] 19. Kang Y-J, Yang W-G, Chae W-S, Kim D-W, Kim S-G, Rotaru H. Administration of 4‑hexylresorcinol increases p53‑mediated transcriptional activity in oral cancer cells with the p53 mutation. Oncol Rep. 2022;48(3):160. pmid:35856441
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref20] 20. Yuan M, Liu X, Wang M, Li Z, Li H, Leng L, et al. A functional variant alters the binding of bone morphogenetic protein 2 to the Transcription Factor NF-κB to regulate bone morphogenetic protein 2 gene expression and chicken abdominal fat deposition. Animals (Basel). 2023;13(21):3401. pmid:37958155
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref21] 21. Ribeiro RC, Sandrini F, Figueiredo B, Zambetti GP, Michalkiewicz E, Lafferty AR, et al. An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma. Proc Natl Acad Sci U S A. 2001;98(16):9330–5. pmid:11481490
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Wasserman JD, Novokmet A, Eichler-Jonsson C, Ribeiro RC, Rodriguez-Galindo C, Zambetti GP, et al. Prevalence and functional consequence of TP53 mutations in pediatric adrenocortical carcinoma: a children’s oncology group study. J Clin Oncol. 2015;33(6):602–9. pmid:25584008
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref23] 23. Varley JM. Germline TP53 mutations and Li-Fraumeni syndrome. Hum Mutat. 2003;21(3):313–20. pmid:12619118
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref24] 24. Kotler E, Shani O, Goldfeld G, Lotan-Pompan M, Tarcic O, Gershoni A, et al. A systematic p53 mutation library links differential functional impact to cancer mutation pattern and evolutionary conservation. Mol Cell. 2018;71(5):873. pmid:30193102
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Mullany LK, Wong K-K, Marciano DC, Katsonis P, King-Crane ER, Ren YA, et al. Specific TP53 mutants overrepresented in ovarian cancer impact CNV, TP53 activity, responses to nutlin-3a, and cell survival. Neoplasia. 2015;17(10):789–803. pmid:26585234
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Masciari S, Dewanwala A, Stoffel EM, Lauwers GY, Zheng H, Achatz MI, et al. Gastric cancer in individuals with Li-Fraumeni syndrome. Genet Med. 2011;13(7):651–7. pmid:21552135
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref27] 27. Ruijs MWG, Verhoef S, Rookus MA, Pruntel R, van der Hout AH, Hogervorst FBL, et al. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes. J Med Genet. 2010;47(6):421–8. pmid:20522432
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref28] 28. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2. Nature. 1997;387(6630):299–303. pmid:9153396
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Alarcon-Vargas D, Ronai Z. p53-Mdm2--the affair that never ends. Carcinogenesis. 2002;23(4):541–7. pmid:11960904
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. Chen J. The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harb Perspect Med. 2016;6(3):a026104. pmid:26931810
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. Wagner J, Portwine C, Rabin K, Leclerc JM, Narod SA, Malkin D. High frequency of germline p53 mutations in childhood adrenocortical cancer. J Natl Cancer Inst. 1994;86(22):1707–10. pmid:7966399
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Kratz CP, Villani A, Nichols KE, Schiffman J, Malkin D. Cancer surveillance for individuals with Li-Fraumeni syndrome. Eur J Hum Genet. 2020;28(11):1481–2. pmid:32814850
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref33] 33. Freed-Pastor WA, Prives C. Mutant p53: one name, many proteins. Genes Dev. 2012;26(12):1268–86. pmid:22713868
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref34] 34. Prives C, White E. Does control of mutant p53 by Mdm2 complicate cancer therapy? Genes Dev. 2008;22(10):1259–64. pmid:18483214
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref35] 35. Willis A, Jung EJ, Wakefield T, Chen X. Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes. Oncogene. 2004;23(13):2330–8. pmid:14743206
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref36] 36. Chen X, Zhang T, Su W, Dou Z, Zhao D, Jin X, et al. Mutant p53 in cancer: from molecular mechanism to therapeutic modulation. Cell Death Dis. 2022;13(11):974. pmid:36400749
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref37] 37. Zhang C, Liu J, Xu D, Zhang T, Hu W, Feng Z. Gain-of-function mutant p53 in cancer progression and therapy. J Mol Cell Biol. 2020;12(9):674–87. pmid:32722796
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref38] 38. Muller PAJ, Vousden KH. p53 mutations in cancer. Nat Cell Biol. 2013;15(1):2–8. pmid:23263379
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref39] 39. Inoue K, Kurabayashi A, Shuin T, Ohtsuki Y, Furihata M. Overexpression of p53 protein in human tumors. Med Mol Morphol. 2012;45(3):115–23. pmid:23001293
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref40] 40. Wang Z, Burigotto M, Ghetti S, Vaillant F, Tan T, Capaldo BD, et al. Loss-of-function but not gain-of-function properties of mutant TP53 are critical for the proliferation, survival, and metastasis of a broad range of cancer cells. Cancer Discov. 2024;14(2):362–79. pmid:37877779
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref41] 41. Jeffers JR, Pinto EM, Rehg JE, Clay MR, Wang J, Neale G, et al. The common germline TP53-R337H mutation is hypomorphic and confers incomplete penetrance and late tumor onset in a mouse model. Cancer Res. 2021;81(9):2442–56. pmid:33637564
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Cells and cell culture

Stable expression mutant p53 genes

Cell growth rate and GFP expression detection by flow cytometry

Subcellular fractionation and immunoblotting

RT-PCR analysis

DNA binding assay

Transcriptional activity assay

CHIP-PCR assay

Statistics

Ethical statement

Results

Detection of p53 T253I germline mutation in ACC patient

Stable expression of clinically observed p53 mutants

T253I shows abnormal autoregulatory feedback with MDM2

Canonical p53 DNA damage responses are negatively impacted in p53 T253I cells

Discussion

Supporting information

S1 Table. An itemized list of antibodies used in this manuscript.

S1 Fig. Histological staining of tumor tissue section shows classical signs of ACC as well as suggests p53 abnormalities.

S2 Fig. Complementation of HEK p53-/- cells with p53 WT-GFP.

S3 Fig. Location of incorporation of p53 mutants into HEK293 cells.

S4 Fig. Steady state expression levels of p53 and MDMD2 in HEK293 p53-/- cells complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53.

S5 Fig. Timecourse of recovery after cisplatin treatment shows maximal induction window for p53-dependent DNA damage responses.

S6 Fig. Luciferase signal strength from luc2P/p53 RE and hRluc/CMV reporters.

Acknowledgments

References

S2 Fig. Complementation of HEK p53^-/- cells with p53 WT-GFP.

S4 Fig. Steady state expression levels of p53 and MDMD2 in HEK293 p53^-/- cells complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53.