Expression of full-length p53 and its isoform Deltap53 in breast carcinomas in relation to mutation status and clinical parameters

Abstract

Background: The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity.

Results: In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Deltap53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Deltap53 have been predicted. We confirmed the expression of Deltap53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Deltap53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53.

Conclusion: Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors, both isoforms show strong correlations in different mutation-dependent mRNA expression patterns.

Figure 1

Schematic representation of the full-length p53 and the alternative splice form Δp53. (A) The mRNA structure of exons VI to X of the full-length p53 and the Δp53 isoform are shown. The removed sequence in Δp53 is located in parts of exon VII, in exon VIII, and in a fraction of exon IX. The alternative splice cassette junction sequence, represented twice in the full-length p53 and once in the alternative splice form, is indicated in red. (B) Structural organization of the full-length p53 and Δp53 and its functional domains. p53 protein domain classification and their locations along the protein according to Swiss-Prot/TrEMBL [12]. Subdomains of main structures are indicated with various colors. Red lines mark the part eliminated by the splicing process of Δp53 and covering aa 257 to 322 of the DNA-binding domain and the complete non-structured spacer region with bipartite nuclear localization signal.

Figure 2

Predicted structural organization of the p53 and Δp53 core domains illustrated by three-dimensional models. Illustrated are CPH predicted models [39] of the p53 and Δp53 isoforms using the RasMol program. The p53 core domain of 204 aa (total length of wild-type p53 is 393 aa) is predicted from aa 94 to 297 with a prediction identity of 100% (557.0 bits score) and the Δp53 core domain is predicted from aa 94 to 274 (total length of Δp53 is 327 aa) with a 93.4% identity and 451.5 bits score (protein position 274 in the Δp53 accordingly corresponds to protein position 340 in the full-length p53). Models and prediction structures for p53 (A) and Δp53 (B) are shown, and the variations are colored by secondary structures as follows: alpha helices in magenta, beta sheets in yellow, turns in pale blue, and all other residues are colored white. Differences between the isoform predictions are indicated with arrows and the N-terminal starting and C-terminal end points are marked in the figure. Due to the alternative splicing a major alpha-helical structure is missing in Δp53 (in A red arrow) and the tertiary protein structure of Δp53 is slightly more compact, as can bee seen from the moved alpha helix, indicated by green arrows. Below: uploaded protein core for the three-dimensional structure prediction query and received structural template.

Figure 3

Kaplan-Meier plot of survival rates for patientswith mutated and unmutated full-length p53 and Δp53. Cumulative breast cancer survival for a subset of patients (50) is shown for three groups of patients, depending on their mutational status of p53 versus Δp53: patients with wild-type Δp53 and wild-type p53 (Wt Δp53 – Wt p53; n = 24), "mutational hybrid" patients with non-mutated (wild-type) Δp53 and mutated full-length p53 (Wt Δp53 - M p53; n = 7), and patients with mutations in Δp53 and p53 (M Δp53 – M p53; n = 19); the significance is p = 0.0498.

Figure 4

p53 and Δp53 standard curve by qRT-PCR. Standard curve plotting showing CO (concentration) in log scale versus Ct (Cycle threshold). The fluorescence signal of the reporter dye (FAM) subtracted by the baseline signal of the passive reference dye (ROX) results in a ratio defined as the normalized reporter signal ΔRn. ΔRn increases with accumulating PCR cycles until it reaches a plateau. Ct represents the fractional cycle number at which significant increase in Rn above a baseline signal of the passive reference dye (ROX) can be detected. Standard curve points are based on serially diluted cDNAs of a mixture of 10 human cancer cell lines in a 1,5 orders of linear dynamic range. All samples were performed in triplets. Red quadrates illustrate data for p53 standard curve; blue squares show data for Δp53 standard curve of the same template. The red line linear represents regression of the standard quantity and the C_T value for Δp53 and green line stand for linear regression of the standard quantity and the C_T value for p53. The comparative Ct value between the two standard curves is 1.40.

Figure 5

Correlation between mRNA expression level of full-length p53 and Δp53 in relation to different molecular breast cancer subtypes in A. wild-type p53 tumors or B. p53-mutated tumors. Both wild-type samples (A) and mutated samples (B) show a wide range of mRNA expression in a.u. (arbitrary units) with significant association to molecular breast cancer subtypes. Note that the spreading is different in the two groups with a more continuously spreading in the wild-type tumors compared to the mutated onces. Samples are marked by their molecular subtype characteristics: Luminal A (dark blue), Luminal B (light blue), ERBB2 (red), Basal (pink), Normal-like (green) and without information (black). Horizontal lines illustrate borders between the quartiles for wild-type (25% = 0.452; 50% = 0.754 and 75% = 1.022) or mutants (25% = 0.569; 50% = 0.956 and 75% = 1.584). The regression line for all samples is drawn with an equation y = 0.789x + 0.100 and a regression coefficient of 0.86 for wild-type and y = 0.973x - 0.049 and a regression coefficient of 0.85 for the mutant samples.

Figure 6

Correlation between mRNA expression level of full-length p53 versus Δp53 in breast carcinomas with various p53 mutations. p53 and Δp53 mRNA expression levels of mutated p53 in human breast tumors are shown with p53 relative mRNA expression in a.u. (arbitrary units) on x-axis and Δp53 on y-axis. Different mutation types are indicated by various symbols. "Mutational hybrids", mutations represented on full-length p53, but removed in Δp53, are marked with open symbols. Mutations present in both isoforms are specified with filled symbols. The shape of the symbols indicate the various mutation types: ■, □ missense mutations, ◊ in frame mutations, ▲, Δ nonsense mutations, * frame shift mutations, ● splice mutations, - mutations in the splice cassette (the two samples full-filling this criteria are highlighted with their sample ID). Horizontal lines show borders between the median values of the relative mRNA expression subgroups: MII vs Wt vs MI. The regression line for missense mutations is drawn with an equation y = 1.03x - 0.12 and a regression coefficient of 0.89. (¹ low p53 value in this sample might be due to mutation in p53 primer binding site, ² low Δp53 value in this sample might be due to mutation in Δp53 primer binding site – for details see Additional file 2).

Figure 7

Histogram of p53 and Δp53 relative mRNA expression. Mean and SEM of the relative p53 and Δp53 mRNA expression in a.u. (arbitrary units) for the different mutation classes: missense, in frame, nonsense, frame shift, splice or splice cassette (splice cass). p53 and Δp53 mRNA expression levels are proportion adjusted according to the comparative Ct of 2.64. The number of cases (n) is given by ¹ for p53 and by ² for Δp53, respectively. Differences in case numbers for p53 and Δp53 are due to some mutations which are located inside the area, but removed by the alternative splicing process of Δp53. The expression level for both full-length p53 and Δp53 between the various mutational classes were highly significant (Table 2).