The function of a heterozygous p53 mutation in a Li-Fraumeni syndrome patient

Abstract

p53 is one of the most extensively studied proteins in cancer research. Mutations in p53 generally abolish normal p53 function, and some mutants can gain new oncogenic functions. However, the mechanisms underlying p53 mutation-driven cancer remains to be elucidated. Our study investigated the function of a heterozygous p53 mutation (p.Asn268Glufs*4) in a Li-Fraumeni syndrome (LFS) patient. We used episomal technology to perform somatic reprogramming, and used molecular and cell biology methods to determine the p53 mutation levels in patient-originated induced pluripotent stem (iPS) cells at the RNA and protein levels. We found that p53 protein expression was not increased in this patient's somatic cells compared with those of a healthy control. p53 mutation facilitates the proliferation of tumor cells by inhibiting apoptosis and promoting cell division. It can inhibit the efficiency of somatic reprogramming by inhibiting OCT4 expression during reprogramming stage. Moreover, not all p53 mutant iPS cell lines have mutant p53 RNA sequences. A small percentage of mutant p53 mRNA is present in the somatic cells from the patient and his mother. In summary, this p53 mutation can promote tumor cell proliferation, inhibit somatic reprogramming, and exhibit random p53 allelic expression of heterozygous mutations in the patient and iPS cells which may be one of the reasons why the people with p53 mutations develop cancer at random. This finding suggested that mutant p53 allelic expression should be added to the risk forecasting of cancer.

Fig 1. Identification of a Asn268Glufs*4 mutation of p53 in a LFS patient.

a-b. The patient was first diagnosed with composite ACC and neuroblastoma at the age of 6 months. Relapse of ACC was diagnosed when he was 16 months old. CT of the mass arising from the left adrenal gland at initial presentation (red arrow) and in the right adrenal gland at relapse (blue arrow). Histologic appearance (H&E staining) of the adrenocortical carcinoma at diagnosis and relapse stage. c. No expression of p53 in the left adrenocortical carcinoma cells from the patient. d. Sanger sequencing of the patient and his mother. The mutation site of p53 is indicated by the red arrow. The p53 sequence is C.801 dup G on chromosome 17 in the patient and his mother. e The domain structure of full-length p53 consisting of an N-terminal transactivation domain (TAD), followed by a proline-rich region (PRR), a central DNA-binding domain (p53C), a tetramerization domain (TET), and an extreme C-terminus (CT)The p53 mutant position of the patient is indicated by the red arrow.

Fig 2. p.Asn268Glufs*4 mutation of p53 loses some functions of wild type p53.

a. Western blotting (WB) of expression of p53, BCL-2, and PUMA in p53^-/- MEF transfected with lentiviruses-mediated p53 WT (WT), mutant (Mut), or an empty vector (EV) control. Arrow, WT p53; arrow head, p53 mutant. b. FACS analysis of apoptosis at Day 3 in the cells from a. * p < 0.05. c. Cell proliferation analysis. d. WB of γH2AX-139 expression. e. Quantitative analysis of γH2X-S139 protein expression in d.

Fig 3. The p53 p.Asn268Glufs*4 mutation inhibits iPS cell generation.

a. WB of p53 protein levels. Mononuclear cells in healthy people with the same age as the patient were used as a control. Arrow, WT p53; arrow head, p53 mutant. b. iPS colony numbers per 1 × 10⁶ monocyte cells used to generate iPSCs at Day 14 after transduction. ***, p < 0.001. c. The percentage of iPS cell lines established on Day 16 after transduction. **, p < 0.01. d. Chromosome numbers in three patient iPS cell lines. e. iPS colony numbers following introduction of WT p53, mutant p53 and vector into p53^+/+ and p53^-/- MEFs were counted on reprogramming Day 14 after transduction. f. Real-time PCR (RT-PCR) of expression of OCT4 in cells following introduction of WT p53 and mutant p53 compared with vector control at the indicated reprogramming time points.

Fig 4. The p53 mutation causes random allelic expression in heterozygous iPS cell lines.

a. Sanger DNA sequencing of three patient iPS cell lines. **, p< 0.01. *, p< 0.05. b. RT-PCR of expression of p53 in iPSCs derived from an LFS patient compared with H1 cells. c. WB of p53 protein expression in iPSCs derived from an LFS patient compared with H1 cells. Arrow, WT p53; arrow head, p53 mutant. d. p53 cDNA sequence from three LFS patient-derived iPS cell lines. e. p53 cDNA sequence from the somatic cells of the patient and his mother.