Common activities and predictive gene signature identified for genetic hypomorphs of TP53

Abstract

Missense mutations that inactivate p53 occur commonly in cancer, and germline mutations in TP53 cause Li Fraumeni syndrome, which is associated with early-onset cancer. In addition, there are over two hundred germline missense variants of p53 that remain uncharacterized. In some cases, these germline variants have been shown to encode lesser-functioning, or hypomorphic, p53 protein, and these alleles are associated with increased cancer risk in humans and mouse models. However, most hypomorphic p53 variants remain un- or mis-classified in clinical genetics databases. There thus exists a significant need to better understand the behavior of p53 hypomorphs and to develop a functional assay that can distinguish hypomorphs from wild-type p53 or benign variants. We report the surprising finding that two different African-centric genetic hypomorphs of p53 that occur in distinct functional domains of the protein share common activities. Specifically, the Pro47Ser variant, located in the transactivation domain, and the Tyr107His variant, located in the DNA binding domain, both share increased propensity to misfold into a conformation specific for mutant, misfolded p53. Additionally, cells and tissues containing these hypomorphic variants show increased NF-κB activity. We identify a common gene expression signature from unstressed lymphocyte cell lines that is shared between multiple germline hypomorphic variants of TP53, and which successfully distinguishes wild-type p53 and a benign variant from lesser-functioning hypomorphic p53 variants. Our findings will allow us to better understand the contribution of p53 hypomorphs to disease risk and should help better inform cancer risk in the carriers of p53 variants.

Keywords: NF-kB; cancer; gene signature; mutant p53; p53.

Fig. 1.

p53 hypomorphs adopt a mutant conformation and express high levels of RRAD. (A) IPA analysis showing significance and effect on regulators with their targets significantly enriched among the genes differentially expressed in p53 hypomorphs P47S and Y107H. The bars represent activation Z-score of the regulators predicted by IPA based on direction of target changes, color represents significance P value, and the number after the bar represents the number of targets affected. (B) Log₂ fold change plotted against –Log₁₀(P-value) for genes that respond to Nutlin-3a treatment after 24 h in WT. Highlighted pink are the genes that are activated to a greater extent in WT than in Y107H in response to treatment and labelled with names are the genes that behave similarly in P47S. The purple labels correspond to known p53 target genes. (C–E) LCL cell lines expressing hypomorphic p53 either (C) P47S—homozygous and heterozygous, (D) Y107H heterozygous, or (E) R273H heterozygous compared to regionally/family matched WT p53 LCL cell lines were treated with 10 μM Nutlin-3a for the indicated time points and harvested. Whole cell lysates were subjected to western blot analysis for the indicated antibodies. (F) WT, P47S, and Y107H HCT116 cells were analyzed for mutant p53 (pAb240) conformation by immunofluorescence analysis. (Scale bar, 25 μm.) (G) Quantification of the fraction of cells staining for mutant conformation (pAb240). Fractions indicated are such that 1.0 = 100%. Data are presented as mean ± SD. n = 25 random fields of view (>250 cells) from each of 2 independent experiments. ^***P < 0.001; ^#P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparisons test. (H) Primary MEFs generated from WT, P47S, and Y107H mice were used to assess mutant p53 conformation by p53-Hsc70 interaction using PLA. Representative images are shown for each condition (Scale bar, 20 μm.) White boxes denote the enlarged insets. (I) Quantification of the average number of PLA signals per cell. Values are expressed as mean ± SD. n = 6 random fields of view (>100 cells) from each of two independent experiments. ^****P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Fig. 2.

p53 hypomorphs exhibit elevated NF-κB signaling. (A and B) LCL cell lines expressing WT or hypomorphic p53, either (A) P47S or (B) Y107H were treated with 0.5 ng/mL TNF-α for the indicated periods. RNA was extracted and analyzed by Q-RT-PCR with primers specific for IL-6 and IL-8 mRNA. Values were normalized for GAPDH mRNA in the same sample. Values are expressed as mean ± SD. Data are representative of n = 6 technical replicates from two independent experiments. ^****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Representative immunohistochemical images of colon, spleen, thymus, and liver sections obtained from 10-wk-old male mice of the indicated genotypes. Tissue sections were stained for total p65 (colon) or for phospho-p65 NF-κB (Ser276). (Scale bar, 50 μm.) (D) Hsc70 immunoprecipitation in colon tissue harvested from 10-wk-old male mice of the indicated genotypes. Immunoprecipitation of Hsc70 or IgG negative control was followed by immunoblotting for p53. (E) Whole cell lysates prepared from the colon and spleen of 10-wk-old male mice of the indicated genotypes were analyzed by western blotting with the indicated antibodies. (F and G) WT and Y107H LCLs were treated with 0.5 ng/mL TNF-α for 0, 1, or 24 h and subjected to ChIP with (9) p65 NF-κB or (9) p53 antibodies, followed by Q-RT-PCR analysis with primers specific to the NF-κB site of the IL-6 and IL-8 promoters or a gene desert (negative control). Values are presented as percentage of input ± SD. The data represent three technical replicates and are representative of two biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 as determined by two-tailed Student’s t test.

Fig. 3.

Altered biochemical properties of hypomorphic p53 proteins. (A) WT, P47S, and Y107H LCLs were treated with or without 1 mM BMH for 30 min followed by immunoblotting for p53. (B) WT, P47S, and Y107H LCLs were treated with vehicle or 1 μg/mL ATO for 16 h and then fixed with or without 1 mM BMH for 30 min followed by immunoblotting for p53. (C) WT or Y107H LCLs were untreated or treated with 10 μM Nutlin-3a (Nut), 1 μM doxorubicin (Dox), or 1 μg/mL ATO for 24 h. Whole-cell lysates were analyzed by western blotting with the indicated antibodies. (D) Cytosolic and nuclear fractions isolated from WT, P47S, and Y107H LCLs were analyzed by western blotting with the indicated antibodies. Histone H3 and GAPDH served as nuclear and cytosolic controls, respectively, to confirm efficiency of subcellular fractionation. (E and F) Chromatin-bound proteins were extracted from nuclear pellets isolated from WT, (E) P47S, and (F) Y107H LCLs using sequentially increasing salt concentrations (0 to 500 mM NaCl) and analyzed by western blotting for p53.

Fig. 4.

p53 hypomorphs share a predictive gene signature. (A) Classification scores for training set samples obtained through leave one out cross-validation based on total 143 unique genes. Probability of 0.5 is used as the classification threshold to calculate accuracy. (B) Classification scores for independent test set. (C) IPA analysis showing enrichment and effect on regulators with targets significantly enriched among the 143 genes from the classifier. (D) Western blot analysis of WT, R175C, and Y220H LCLs treated with 10 μM Nutlin-3a for 24 h. (E) Western blot analysis of WT, P47S, G360A, E11Q, and R110H LCLS treated with 10 μM Nutlin-3a for 24 h.