p53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo

Abstract

Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/- tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data - while cautioning that not all genetic mouse models faithfully represent the human situation - demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.

Figure 1

Analysis of the databases of sporadic human breast cancer (a) and sarcomas (b) (METABRIC, provisional TCGA and others, see Materials and Methods section) show a high frequency of wtp53 allele loss (LOH) in missense mutp53 tumors

Figure 2

Survival curves analyzing tumor onset of sarcomas and breast carcinomas in p53Q/+Neu, p53−/+Neu and p53+/+Neu mouse cohorts. (a) Sarcoma onset is faster in p53Q/+Neu compared with p53−/+Neu mice. This indicates either a DN effect over wtp53 or, alternatively, p53 LOH resulting in mutp53 GOF specifically in sarcoma. (b) Breast cancer latency in p53Q/+Neu and p53−/+Neu siblings is similar, reflecting that the majority of p53Q/+ breast tumors did not undergo LOH (see Figure 3b) in contrast to human breast cancer, and also did not exert a DN effect over wtp53 but simply behaved as a LOF allele. Kaplan–Meier analysis;n, number of mice; P, log rank statistics

Figure 3

Loss of wtp53 allele is required for missense mutant p53 stabilization and GOF. (a) The vast majority of sarcomas (16/17 cases, 94%) have stabilized mutp53. In contrast, the majority of breast carcinomas (16/20 cases, 80%) do not. Immunohistochemistry for mutp53. Mouse identity in parentheses. Arrows indicate the osteoid in osteosarcoma. (b) Analysis of wtp53 copy number in sarcomas and breast carcinomas of p53Q/+Neu mice by quantitative genotyping. Tumors with mutp53 stabilization (all sarcomas and three breast cancers tested) have significantly higher LOH than tumors without mutp53 stabilization (majority of breast cancers). Note, as sarcomas have high normal stroma contamination (top, blue mutp53-negative stromal cells, which do not have LOH), the actual LOH in sarcomas is most likely even higher because of dilution of the tumor genotype, causing LOH underestimation. For the same reason, copy numbers of the two highest sarcoma cases (two left red bars) are likely inflated. The wtp53 signal was normalized to the Rosa26 signal. Tail biopsies from p53+/+ (two wt alleles), p53Q/+, p53−/+ (one wt allele) and p53−/− mice (no wt alleles) were used as normal control tissues without LOH. Bars represent mean±S.D. of two technical replicas of individual cases. ***P<0.001. (c) Schematic diagram of the proposed mechanism for mutp53 stabilization and GOF in heterozygous tumors. Loss of the wtp53 allele (LOH) causes accumulation of highly stabilized mutp53 protein, which triggers tumor development and is the principle mechanism and prerequisite of GOF

Figure 4

Real-time qPCR analysis of wtp53 target genes Mdm2, p21, Bax and Puma shows that their expression is largely decreased in samples with mutp53 stabilization compared with samples without mutp53 stabilization