Differential Gain-of-Function Activity of Three p53 Hotspot Mutants In Vivo

Abstract

The majority of TP53 missense mutations identified in cancer patients are in the DNA-binding domain and are characterized as either structural or contact mutations. These missense mutations exhibit inhibitory effects on wild-type p53 activity. More importantly, these mutations also demonstrate gain-of-function (GOF) activities characterized by increased metastasis, poor prognosis, and drug resistance. To better understand the activities by which TP53 mutations, identified in Li-Fraumeni syndrome, contribute to tumorigenesis, we generated mice harboring a novel germline Trp53R245W allele (contact mutation) and compared them with existing models with Trp53R172H (structural mutation) and Trp53R270H (contact mutation) alleles. Thymocytes from heterozygous mice showed that all three hotspot mutations exhibited similar inhibitory effects on wild-type p53 transcription in vivo, and tumors from these mice had similar levels of loss of heterozygosity. However, the overall survival of Trp53R245W/+ and Trp53R270H/+ mice, but not Trp53R172H/+ mice, was significantly shorter than that of Trp53+/- mice, providing strong evidence for p53-mutant-specific GOF contributions to tumor development. Furthermore, Trp53R245W/+ and Trp53R270H/+ mice had more osteosarcoma metastases than Trp53R172H/+ mice, suggesting that these two contact mutants have stronger GOF in driving osteosarcoma metastasis. Transcriptomic analyses using RNA sequencing data from Trp53R172H/+, Trp53R245W/+, and Trp53R270H/+ primary osteosarcomas in comparison with Trp53+/- indicated that GOF of the three mutants was mediated by distinct pathways. Thus, both the inhibitory effect of mutant over wild-type p53 and GOF activities of mutant p53 contributed to tumorigenesis in vivo. Targeting p53 mutant-specific pathways may be important for therapeutic outcomes in osteosarcoma.

Significance: p53 hotspot mutants inhibit wild-type p53 similarly but differ in their GOF activities, with stronger tumor-promoting activity in contact mutants and distinct protein partners of each mutant driving tumorigenesis and metastasis.

Figure 1.. Similar inhibitory effect among p53 mutations in vivo.

(A) RNA levels of p53 downstream target genes Eda2r, Cdkn1a (p21), Bbc3 (Puma), Bax, and Ccng1 were determined by RT-qPCR 4 hours after 2.5 Gy irradiation in WT (+/+) (N=4 for both non-IR and IR treatment), Trp53^+/− (+/−) (N=3), Trp53^R172H/+ (R172H/+) (N=5), Trp53^R245W/+ (R245W/+) (N=4), Trp53^R270H/+ (R270H/+) (N=4), and Trp53^−/− (−/−) (N=3 for non-IR and N=5 for IR) mouse thymuses. (B) IHC staining of cleaved caspase-3 (CC3) was performed on thymus tissues from mice with the indicated genotypes. Scale bar = 100μm (C) CC3-stained cells were quantified in five random fields. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 by t-test.

Figure 2.. Inhibitory effect of mutant p53 in vivo.

(A) IE of mutant p53 by IR treatment in Trp53^neo/− and Trp53^neo/mut mice. Relative RNA levels of Cdkn1a (p21), Bax, and Bbc3 (Puma) were determined by RT-qPCR in thymuses of 1-month-old Trp53^neo/− (Neo/−), Trp53^neo/R172H (Neo/R172H), Trp53^neo/R245W (Neo/R245W), Trp53^neo/R270H (Neo/R270H), and Trp53^−/− (−/−) mice, 4 hours after 6 Gy IR treatment. At least three mice were used for each genotype. (B) Kaplan-Meier survival curves of Trp53^neo/− (N=48), Trp53^neo/R172H (N=36), Trp53^neo/R245W (N=22), Trp53^neo/R270H (N=23), and Trp53^−/− (N=19) mice. (C) Expression of p53 target genes in Trp53^neo/Mut tumors compared to Trp53^neo/− tumors was determined by RT-qPCR. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 by t-test.

Figure 3.. Distinct tumor phenotypes among mutant p53 mice.

Kaplan-Meier survival curves of Trp53^+/− (N=33), Trp53^R172H/+ (N=35), Trp53^R245W/+ (N=105), and Trp53^R270H/+ (N=38) mice at 22 months (A) and 14 months (B). (C) Loss of heterozygosity analysis in Trp53^R172H/+ (N=31), Trp53^R245W/+ (N=26), and Trp53^R270H/+ (N=14) tumors. (D) Kaplan-Meier survival curves of Trp53^−/− (N=19), Trp53^R172H/R172H (N=34), Trp53^R245W/R245W (N=139), and Trp53^R270H/R270H (N=44) mice. (E) p53 levels were detected by IHC staining in Trp53^R172H/+ (N=19), Trp53^R172H/R172H (N=6), Trp53^R245W/+ (N=16), Trp53^R245W/R245W (N=25), Trp53^R270H/+ (N=20), and Trp53^R270H/R270H (N=13) tumors. Positively stained nuclei were counted by ImageJ software. (F) Kaplan-Meier survival curves of homozygous Trp53^R245W/R245W and Trp53^R270H/R270H mice with 0-40% and 60-100% positive p53 nuclei by IHC staining. * indicates p<0.05, ** indicates P < 0.01, and *** indicates P < 0.001 by t-test.

Figure 4.. Mutant allele-specific differences in tumorigenesis.

(A) Lymphoma-specific survival curves of Trp53^R172H/+ (N=10) and Trp53^R245W/+ (N=9) mice (lymphomas as the only cancer were not observed in Trp53^R270H/+. (B) Female mouse-specific survival curves for the Trp53^R172H/+ (N=19), Trp53^R245W/+ (N=60), and Trp53^R270H/+ (N=20) mice. (C) p53 IHC staining in Trp53^R172H/+, Trp53^R245W/+, and Trp53^R270H/+ osteosarcomas. (D) Percent metastasis observed in Trp53^+/− (0/6), Trp53^R172H/+ (1/8), Trp53^R245W/+ (5/11), and Trp53^R270H/+ (4/10) mice. * indicates P < 0.05 by t-test.

Figure 5.. Different pathways contribute to osteosarcoma metastasis among p53 mutants.

(A) Supervised clustering based on genotype using the Pearson distance and Ward linkage. (B) Principal component analysis of all osteosarcoma RNA-seq data. (C) Upregulated differentially expressed genes (DEGs) for each mutant were identified by DESeq2 through comparing the individual Trp53^Mut/+ tumors to Trp53^+/− tumors. The significance criteria were adjusted P-value < 0.05 and fold change > 2. (D) Immunoprecipitation experiments were performed in primary osteosarcoma cell lines with LOH of the p53 wild type allele (designated as O). Top panel shows input. Ctl: control antibody for IP; p53: CM5 antip53 antibody. N.S.: nonspecific; IP: immunoprecipitation; IB: immunoblot. (E) IPA and EnrichR analysis results of top upstream transcription factors regulating the promoters of genes differentially expressed in 14W Trp53Si-1 cells. Ctl: control siRNA; Si-1 and Si-2: Trp53siRNA.