Amino-terminal p53 mutations lead to expression of apoptosis proficient p47 and prognosticate better survival, but predispose to tumorigenesis

Abstract

Whereas most mutations in p53 occur in the DNA-binding domain and lead to its functional inactivation, their relevance in the amino-terminal transactivation domain is unclear. We show here that amino-terminal p53 (ATp53) mutations often result in the abrogation of full-length p53 expression, but concomitantly lead to the expression of the amino-terminally truncated p47 isoform. Using genetically modified cancer cells that only express p47, we demonstrate it to be up-regulated in response to various stimuli, and to contribute to cell death, through its ability to selectively activate a group of apoptotic target genes. Target gene selectivity is influenced by K382 acetylation, which depends on the amino terminus, and is required for recruitment of selective cofactors. Consistently, cancers capable of expressing p47 had a better overall survival. Nonetheless, retention of the apoptotic function appears insufficient for tumor suppression, because these mutations are also found in the germ line and lead to Li-Fraumeni syndrome. These data from ATp53 mutations collectively demonstrate that p53's apoptosis proficiency is dispensable for tumor suppression, but could prognosticate better survival.

Keywords: ATp53 mutants; acetylation; amino terminus; full-length p53; p47.

Fig. 1.

ATp53 indels lead to loss of p53 expression but result in p47 expression. (A) Sequence alignment of cancer-derived alterations identified within amino acids 1–40. All indels lead to the generation of a stop codon (TGA/TAA) within the first 43 aa. The next methionine (ATG) after the stop signal is underlined in each case. (B and C) p53 null H1299 cells were transfected with selected ATp53 indels and analyzed by immunoblotting with an amino-terminal–specific p53 antibody (DO1) (recognizing amino acids 11–25), or an antibody that recognizes between residues 46–55 (Pab 1801) (B). One representative ATp53 indel, the 97delT, was mutated to substitute its methionines at amino acid 40 and/or 44 to alanine, and the plasmids were transfected similarly to analyze the expression of p47 (C). p53 and p47 (where the first ATG in p53 was mutated to a stop codon) plasmids were used as controls, and representative blots are shown. Arrowheads indicate position of p47 and p53. (D and E) Primary colorectal samples with either DBD-domain mutations (DBD), or amino-terminal mutations (AT) were used for immunoblot analyses with the DO1 antibody or the carboxyl-terminal–specific PAb421 antibody (recognizing amino acids 371–380) (D). RKO^+/+ and RKO^−/− cells treated with etoposide were used as positive controls for p53 and p47 expression, respectively (see Fig. 2 for details). Arrowheads indicate position of p47 and p53, and asterisk represents nonspecific bands. Lo and hi refer to low and higher exposures of the blots. Three of the samples, one representing a DBD mutation (3616: p.R175H), and two representing the amino-terminal mutations (3431: c.97_115del; 3624: frameshift) were used for immunohistochemical analysis with the pan-p53 (CM1) or amino-terminal specific (DO1) antibodies (E). Representative pictures from lower (Top) and higher (Bottom) magnification are shown.

Fig. 2.

Endogenous p47 is induced by multiple stimuli. (A and B) Immunoblot analysis of p47 and p53 induction upon treatment with various stimuli was determined by immunoblotting using total cell lysates from the respective RKO^−/− and RKO^+/+ cells and a pan-p53 antibody (CM1) or the DO1 antibody (A). Half-life of p47 and p53 was determined in these cells with or without etoposide treatment for 24 h, followed by cycloheximide (CHX) treatment for the indicated time periods (B). Etop, etoposide; HU, hydroxyurea; -serum, serum starved for 24 h; TG, thapsigargin; UT, untreated. Arrowheads indicate position of p47 and p53. Numbers below the blots indicate quantification of p53/p47, relative to untreated or at time 0 h. (C) RKO^−/− and RKO^+/+ cells with or without etoposide treatment were used for immunofluorescence analysis using the DO1 antibody (Top), or the pan-p53 CM1antibody (Bottom). DAPI stain highlight nucleus.

Fig. 3.

p47 contributes to apoptotic target gene activation and cell death. (A) Various p53 target genes expression was analyzed by quantitative real-time PCR after treatment of RKO^−/− and RKO^+/+ cells with etoposide for the indicated time periods. Relative expression was normalized against gapdh expression. (B–D) Cells were transfected with control (pSuper) or Shp53 and treated with etoposide or cisplatin (CDDP) and used for immunoblot analysis (B), or for real-time PCR analysis of the indicated genes (C). Cell death was determined concurrently after 48 h after treatment (D). Arrowheads indicate position of p47 and p53 in B. All experiments are representative of at least three independent experiments, and data are represented as mean ± SDs.

Fig. 4.

p47 is capable of inducing cell death and selectively activates apoptotic target genes. (A and B) The indicated constructs were transfected into H1299 cells, and cell death was analyzed 24 h later by flow cytometry, following propidium iodide/Annexin V staining (A), or by sub-G₁ analysis (B). Representative data are shown. (C) H1299 and Saos2 cells were transfected with the indicated plasmids and selected for 8–14 d, and cellular colonies were stained with crystal violet and visualized. Representative data from at least three independent repeats are shown. (D and E) The indicated plasmid constructs were transfected into p53-deficient H1299 cells together with the indicated p53-target promoters linked to the luciferase gene, and luciferase activity was determined (Left). Immunoblots indicate expression levels of the transfected proteins (Right) (D). Real-time quantitative PCR of endogenous target genes were analyzed 24 h after transfection of the indicated p53/p47 constructs in H1299 cells. Relative expression was normalized against gapdh expression (E). Experiments are representative of at least three independent repeats, and SDs are shown.

Fig. 5.

K382 acetylation is required for efficient p21 gene activation, but dispensable for apoptotic target gene activation. (A and B) Posttranslational modifications on endogenous p47 and p53 after cellular stimulation with indicated reagents were analyzed by immunoblotting using respective antibodies. PACL4 is a pancreatic cell line with p300 deletion and with a mutant p53 (B). Arrowheads indicate position of p47 and p53, and asterisks represents nonspecific bands. (C and D) H1299 cells were transfected with the indicated p53 mutant constructs, and cell lysates (C) or mRNA (D) were used for immunoblot or quantitative PCR analysis of target genes, respectively. (E) Chromatin immunoprecipitation was performed by using anti-TAF1, hCAS1, or IgG control antibodies, followed by PCR amplification of p21 (sites p21A and p21B) or Aip1 promoter regions, from RKO^−/− and RKO^+/+ cells with and without etoposide treatment. Input is from genomic DNA without chromatin immunoprecipitation. Asterisk represents primer dimers. (F and G) H1299 cells were transfected with the indicated p53 constructs in various combinations with the p47 plasmid, and K382 acetylation was determined by immunoblotting (F). Asterisk represents nonspecific band. mRNA from parallel cultures were used for quantitative PCR analysis of p21 (G). All experiments are representative of at least three independent experiments, and data are represented as mean ± SDs.

Fig. 6.

ATp53 mutations lead to Li–Fraumeni syndrome. (A) Quantification of somatic or germ-line mutations within the various regions of the p53 gene in human cancers is shown. Data were analyzed from IARC database for p53 mutations. (B and C) Characteristics of French LFS families with ATp53 mutations leading to stop codon within amino acid 40 region is shown (B). Pedigree of family 36 is shown (C). Respective age of patients and cancer types are indicated.

Fig. 7.

Clinical effects of ATp53 alterations. (A) Schematic shows p53 and p53EII transcripts (Top). H1299 cells were transfected with the indicated constructs, with either the G or C polymorphism in codon 72, and analyzed by immunoblotting with pan-p53 polyclonal CM1 antibody. (B and C) p53EII mRNA levels were analyzed by quantitative real-time PCR in colorectal samples. Relative levels to gapdh are shown for a subset of samples, to highlight the p53EII^Lo (relative levels <1.5) and the p53EII^Hi (relative levels >1.5) groups (B). Kaplan–Meier plots of overall survival of patients from this cohort are shown. (C, Top). Table shows mean survival (in months) of patients with p53EII^Hi (relative levels >2.0), p53EII^Lo, or with ATp53 indels (C, Bottom). (D and E) Model for p47 generation by ATp53 indels and its role in regulation of cell death. ATp53 indels generate a stop signal (indicated by “xxxx”) and, thus, disrupt the expression of full-length p53. Translation from the next methionine therefore leads to the generation of the p47 isoform (D). Under naïve conditions, p47 is generally abundant and nuclear, and is found to be bound onto target promoters, although target genes are not activated. By contrast, p53 is a labile protein that is degraded, and is minimally bound to target promoter DNA (E, Top). Upon stimulation by various stress agents, p47 is activated, leading to accumulation without any increase in protein stability. However, p47 is not acetylated at K382 because of lack of the amino-terminal domain, and does not recruit TAF1 to targets such as p21, resulting in inefficient p21 expression. Nonetheless, hCAS1 is recruited to apoptotic target promoters such as Aip1, even in the absence of K382 acetylation, thereby leading to their activation. Cellular stimulation, however, leads to stabilization of p53, which is the primary mechanism for increasing its abundance (E, Bottom). Stabilized p53 is acetylated on K382 and is able to recruit TAF1 to arrest target promoters. Concomitantly, p53 is also able to bind to apoptotic target promoters, recruiting hCAS1 and result in their activation. Red dots represent acetylation on K382.