Mutant p53 leads to low-grade IFN-I-induced inflammation and impairs cGAS-STING signalling in mice.

Type I interferons (IFN-Is) are a class of proinflammatory cytokines produced in response to viruses and environmental stimulations, resulting in chronic inflammation and even carcinogenesis. However, the connection between IFN-I and p53 mutation is poorly understood. Here, we investigated IFN-I status in the context of mutant p53 (p53N236S , p53S). We observed significant cytosolic double-stranded DNA (dsDNA) derived from nuclear heterochromatin in p53S cells, along with an increased expression of IFN-stimulated genes. Further study revealed that p53S promoted cyclic GMP-AMP synthase (cGAS) and IFN-regulatory factor 9 (IRF9) expression, thus activating the IFN-I pathway. However, p53S/S mice were more susceptible to herpes simplex virus 1 infection, and the cGAS-stimulator of IFN genes (STING) pathway showed a decline trend in p53S cells in response to poly(dA:dT) accompanied with decreased IFN-ß and IFN-stimulated genes, whereas the IRF9 increased in response to IFN-ß stimulation. Our results illustrated the p53S mutation leads to low-grade IFN-I-induced inflammation via consistent low activation of the cGAS-STING-IFN-I axis, and STAT1-IRF9 pathway, therefore, impairs the protective cGAS-STING signalling and IFN-I response encountered with exogenous DNA attack. These results suggested the dual molecular mechanisms of p53S mutation in inflammation regulation. Our results could be helping in further understanding of mutant p53 function in chronic inflammation and provide information for developing new therapeutic strategies for chronic inflammatory diseases or cancer.

Gain of function in the mouse model of a recurrent mutation p53N236S promotes the formation of double minute chromosomes and the oncogenic potential of p19ARF.

The mutation p53N236S (p53S) has been identified as one of the recurrent mutations in human cancers by TCGA database. Our in vitro data revealed the oncogenic gain of function of p53S. To understand the function of p53S in vivo, we generated the p53S knock-in mouse. The p53S/S mice manifested highly invasive lymphomas and metastatic sarcomas with dramatically increased double minute chromosomes. The survival curve, the incidence of tumors and the tumor spectrum of p53S/S mice is very similar to the p53R172H mouse model. The p53S/+ mice showed delayed onset of tumorigenesis and a high metastasis rate (40%) and low loss of heterozygosity rate (2/16). The activation of CDKN2A pathway in p53S/S MEF and tumors, and the accumulation of p19ARF protein in tumor tissues suggested p19ARF might contribute to the accumulation of mutant p53S protein in the tumor and promote tumorigenesis. The high expression of p19ARF correlated with mutant p53 accumulation and tumor progression, suggesting a dual role of p19ARF in tumor promotion or suppression that might depend on the p53 mutation status in tumor cells. The oncogenic gain of function of this recurrent mutation p53S prompts the reconsideration of p53 mutations function that occurs at a low frequency.

p53N236S Activates Autophagy in Response to Hypoxic Stress Induced by DFO.

Hypoxia can lead to stabilization of the tumor suppressor gene p53 and cell death. However, p53 mutations could promote cell survival in a hypoxic environment. In this study, we found that p53N236S (p53N239S in humans, hereinafter referred to as p53S) mutant mouse embryonic fibroblasts (MEFs) resistant to deferoxamine (DFO) mimic a hypoxic environment. Further, Western blot and flow cytometry showed reduced apoptosis in p53S/S cells compared to WT after DFO treatment, suggesting an antiapoptosis function of p53S mutation in response to hypoxia-mimetic DFO. Instead, p53S/S cells underwent autophagy in response to hypoxia stress presumably through inhibition of the AKT/mTOR pathway, and this process was coupled with nuclear translocation of p53S protein. To understand the relationship between autophagy and apoptosis in p53S/S cells in response to hypoxia, the autophagic inhibitor 3-MA was used to treat both WT and p53S/S cells after DFO exposure. Both apoptotic signaling and cell death were enhanced by autophagy inhibition in p53S/S cells. In addition, the mitochondrial membrane potential (MMP) and the ROS level results indicated that p53S might initiate mitophagy to clear up damaged mitochondria in response to hypoxic stress, thus increasing the proportion of intact mitochondria and maintaining cell survival. In conclusion, the p53S mutant activates autophagy instead of inducing an apoptotic process in response to hypoxia stress to protect cells from death.

P53 Mutant p53N236S Regulates Cancer-Associated Fibroblasts Properties Through Stat3 Pathway.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play important roles in cancer development and progression. Recent studies show that p53 plays a cell non-autonomous tumor-suppressive role to restrict tumor growth in CAFs. However, the role of p53 mutant in CAFs remains obscure. METHODS: In this study, the contribution of p53 mutant p53N236S (p53S) to CAFs activation was examined using mouse embryonic fibroblasts (MEFs) from wild-type (WT), p53 deficient (p53-/- ) and p53S/S mice. The role of p53S in MEFs in inducing prostate cancer cell growth and metastasis was studied by utilizing xenograft models and transwell assays. The effects of p53S on the properties of CAFs were assessed by measuring CAFs-specific factors expression and functional collagen contraction assay. Moreover, Microarray data were analyzed by GSEA and Stat3 signaling was inhibited to further determine p53S's role in the CAFs activation. RESULTS: We found that p53S/S MEF accelerated cancer cells growth and metastasis compared with WT and p53-/- MEF. We also found that p53S induced significantly increasing collagen contraction in fibroblasts and overexpression of CAFs-specific factors, such as α-smooth muscle actin (α-SMA), FGF10 and CXCL12. p53S regulated these CAF-specific properties through Stat3 activation. CONCLUSION: Our results illustrate that p53S plays an important role in CAFs activation by the Stat3 pathway. The study indicates that cancer cells and fibroblasts interaction promotes prostate cancer cell growth, migration and invasion due to p53S expression in fibroblasts.

p53 Mutant p53N236S Induces Neural Tube Defects in Female Embryos.

The p53 is one of the most important tumor suppressors through surveillance of DNA damages and abnormal proliferation signals, and activation the cell cycle arrest and apoptosis in response to stress. However, the mutation of p53 is known to be oncogenic by both loss of function in inhibiting cell cycle progress and gain of function in promoting abnormal proliferation. In the present study, we have established a knock in mouse model containing an Asn-to-Ser substitution at p53 amino acid 236 by homologous recombination (p53N236S). Other than tumorigenesis phenotype, we found that p53S/S mice displayed female-specific phenotype of open neural tube in brain (exencephaly) and spinal cord (spina bifida). The occurrence rate for embryonic exencephaly is 68.5% in female p53S/S mice, which is much more than that of in p53-/- mice (37.1%) in the same genetic background. Further study found that p53N236S mutation increased neuronal proliferation and decreased neuronal differentiation and apoptosis. To rescue the phenotype, we inhibited cell proliferation by crossing Wrn-/- mice with p53S/S mice. The occurrence of NTDs in p53S/S Wrn-/- mice was 35.2%, thus suggesting that the inhibition of cell proliferation through a Wrn defect partially rescued the exencephaly phenotype in p53S/S mice. We also report that p53S decreased expression of UTX at mRNA and protein level via increasing Xist transcript, result in high female-specific H3K27me3 expression and repressed Mash1 transcription, which facilitating abnormal proliferation, differentiation, and apoptosis, result in the mis-regulation of neurodevelopment and neural tube defects (NTDs).

The transcription and expression profile of p53N236S mutant reveals new aspects of gain of function for mutant p53.

Missense mutations in the p53 coding gene cause loss and gain of function. We have identified a hotspot mutation, p53N236S , which results in the aggressive progression of tumorigenesis in a knock-in mouse model. To understand the biological significance of the p53N236S mutation, we performed ChIP-on-chip combined with microarray assay to profile the regulated gene expression pattern. We could classify the p53N236S mutant function into six categories. Among these, we reveal a new aspect of gain of function, the enhancement of wild-type p53 function, which has not been reported previously. We also show the existence of residual wild-type p53 function in p53N236S . Our data shed light on understanding the difference between this type of low-incidence hotspot p53 mutations and classical hotspot mutations.