3rd p53 Workshop in Japan ’20

organized by Marco M Candeias & Rieko Ohki for MBSJ2020
online on Dec 3rd 2020, from 3:30pm (Japan time)
(also available on-demand until Dec 11)

Register here: MBSJ2020

List of talks:

1- First insights on the regulation and function of the mammalian p53 isoform: Delta160p53

Marco M Candeias 
Kyoto University / INSA

The p53 gene is well conserved among animals, always with a clear role in the response to DNA damage and induction of programmed cell death, a true guardian of the genome. A recent look into the structure of the p53 gene in more than 50different animal species reveals however putative taxon-specific p53 gene products such as the mammalian Delta160p53 isoform and the primate Delta133p53 isoform. During the last 10 years we have been studying the tight regulation Delta160p53 is subjected to as a strong pro-survival and pro-invasive cue in the cell, as well as the different mechanisms by which this regulation becomes impaired in cancer. I will discuss these in light of our most recent findings on the post-translational modification and sub-cellular localization of Delta160p53 and propose a mode of action for Delta160p53 in mammalian cells and cancer.

2- The ubiquitin E3 ligase Huwe1 regulates oocyte maturation and preimplantation embryo development independently of p53

 Manabu Kurokawa
Kent State University 

The tumor suppressor p53 is tightly regulated at the level of protein stability. Over 20ubiquitin E3 ligases have been found to promote the degradation of p53 protein.Although it is known that MDM2 is the major E3 ligase for p53, the contribution of other E3 ligases in regulating p53 remains elusive. HUWE1 is a HECT-domain ubiquitinE3 ligase that was initially discovered as a p53-targeting E3 ligase ubiquitously expressed in various tissues. Here, we investigated the role of HUWE1 in the female reproductive system. Homozygous deletion of Huwe1 in mouse oocytes of primary follicles caused oocyte death and female infertility, whereas acute depletion of HUWE1protein by Trim-Away technology did not impact oocytes from antral follicles.Interestingly, oocytes from Huwe1 heterozygous females matured and fertilized normally, but the majority of embryos that lacked maternal Huwe1 were arrested at the morula stage after fertilization. Consequently, Huwe1 heterozygous females only produced wild-type pups. Surprisingly, concomitant knockout of p53 did not recover fertility of the Huwe1 knockout females, indicating that the functions of HUWE1 in oogenesis and embryonic development are independent of p53. Our findings makeHUWE1 a unique and critical maternal factor indispensable for maintaining the quality of oocytes and embryos.

3- P53 is not indispensable for the restoration of damaged tissues by irradiation in the small-fish model, medaka (Oryzias latipes)

 Takako Yasuda
University of Tokyo 

A tumor suppressor protein p53 has an important role in regulating the induction of cell cycle arrest and apoptosis in response to cellular stress signals to cull irreparable damages for ultimate benefit of the organism. We examined the role of p53 for the restoration of damaged tissues following gamma-ray irradiation in the highly radiosensitive tissues, developing embryonic brain and matured adult testis, of a small fish model, medaka (Oryzias latipes). Medaka offer advantages as a vertebrate model system, since the transparency and small size of its embryos enable clear detection of apoptotic cells in the whole developing brain. We found that, in the irradiated p53-deficient embryonic brain, the diminishment of apoptosis induction could facilitate tissue regeneration after irradiation, whereas the irradiated wild-type embryos retained the structural abnormalities even at the time of hatching. In addition, the simple architecture of testis of medaka enables us a precise and more convenient identification of differentiating spermatogenic cells compared with mammals. In the irradiated p53-deficient testes, the absence of apoptotic induction might trigger transient mis-differentiation of spermatogonial cells into ovum-like cells in the testis(testis-ova). Then, all of the induced testis-ova were eliminated via p53-independent apoptosis and spermatogenesis was restored within 1 month after the irradiation.Collectively, our findings strongly suggest that p53 could not be indispensable for the restoration of irradiation-induced damaged tissues.

4- ZBTB2 links p53-deficiency to HIF-1-mediated hypoxia signaling in cancers

Hiroshi Harada
Kyoto University

Both dysfunction of a tumor suppressor, p53, and aberrant activation of a hypoxia-inducible transcription factor, HIF-1, are known to induce malignant phenotypes and therapy resistance of cancer cells. However, functional and mechanistic relationships between them remain to be elucidated, which makes it difficult to establish rational therapeutic strategies for p53-deficient cancers. Here we conducted a genome-wide expression screening and identified Zinc finger and BTB domain-containing protein 2(ZBTB2) as a novel factor linking the two. We found that ZBTB2 increased the transactivation activity of HIF-1α in functional p53-deficient cancer cells. ZBTB2facilitated growth and invasion of cancer cells
in vitro and promoted the growth of subcutaneous tumor when functional p53 was deficient. Immunohistochemical analysis of clinical cancer samples found ZBTB2 expression levels associated with poor overall survival of patients. Deletion and point mutation analyses revealed ZBTB2 N-terminus, whose importance in ZBTB2 homodimerization was predicted by in silico homology modeling, critical for the ZBTB2 activity. Polypeptides consisting of the ZBTB2 N-terminus competitively inhibited the ZBTB2 homodimer formation and significantly suppressed the ZBTB2-HIF-1 axis. Our data indicates that ZBTB2 promotes growth and malignancy of p53-deficient tumors by activating HIF-1 and provides a rational basis to target the ZBTB2 homodimerization by a mimetic of the ZBTB2 N-terminus for suppressing the ZBTB2-HIF-1 axis in p53-deficient cancers.

5- Gain-of-function mutation of p53 for malignant progression of cancer

Masanobu Oshima 
Kanazawa University 

The accumulation of genetic alterations in driver genes are responsible for the development and malignant progression of colorectal cancer (CRC). Among them, the p53 mutation is found in about 60% of colorectal cancers, and a majority of mutations are missense-type at hot-spots, suggesting an acquired oncogenic role of mutant p53by gain-of-function (GOF) mechanism. We generated Apc Trp53 (R270H) conditional mutant mice, in which GOF mutant p53 was expressed in the intestinal epithelia.Histological analysis indicated that p53 R270H caused increased branching and submucosal invasion of intestinal tumors, while the loss of wild-type p53 (p53 Null)has a limited effect. Furthermore, the GOF mutant p53 promotes metastasis when combined with Kras activation and TGF-beta suppression. Notably, RNAseq analysis indicated that GOF p53 mutation induces activation of NF-kB pathway possibly through increasing promoter accessibility by chromatin remodeling, which may contribute to inflammatory microenvironment generation. In addition to GOF mutation, loss of wild-type p53 by loss of heterozygosity (LOH) is frequently found in malignant human CRC.We thus examined the role of p53 LOH in combination with GOF mutation in malignant progression using mouse tumor organoids (AKTP cells) that carried multiple driver mutations. Notably, AKTP cells with GOF mutation and LOH of p53 led to increased survival and clonal efficiency of cancer cells, and promoted metastasis. These results suggest that p53 GOF mutations together with p53 LOH accelerate metastasis through acquisition of oncogenic abilities and activation of inflammatory pathways.

6- Knockout phenotypes of the p53 family

Gerry Melino
University of Rome / University of Cambridge

The p53-family member p73 plays a key role in the development of the central nervous system, senescence and in tumour formation. Here, we found that TAp73-/- mice show elevated tumour incidence, with solid evidence of metabolic (Cox4i1, GLS, glycine biosynthesis), apoptotic (CD95, Bax), angiogenic (via HIF-1α) regulation. The role of p73 in neuronal differentiation is complex and involves several downstream pathways, including SOX-2, Hey-2, TRIM32, p75NTR, GLS2 and Notch signalling activity as well as the regulation of microRNAs like miR-34a. Accordingly, the p73-/- mice displayed hippocampal dysgenesis. Incomplete results still exist in the relationship between p73 and Alzheimer’s disease and this remains an important area for further experimentation. The p53-family member p63 mediates different cellular responses mainly affecting epithelial biology. In addition to two amino terminal isoforms, TAp63 and DNp63, the 3’ end of p63 mRNA undergoes tissue-specific extensive alternative splicing that leads to several more isoforms, including p63a (containing a Sterile Alpha Motif), p63b and p63g. To test the hypothesis that these exert different functions in vivo, a p63a-targeted allele deleting exon 13 (D13p63) was generated, replacing the p63a isoform with p63b. The heterozygous female mice were completely sterile (controlled by TAp63a), while thymus or skin (controlled byDNp63) were normal. These mice showed premature ovarian failure (POF) due to uncontrolled primary oocyte cell death. These results directly implicate uncontrolled p63 splicing and/or mutation as a major cause of POF, contributing to expanding the knowledge of the genetic abnormalities of female infertility.

7- A novel p53 downstream tumor promotion pathway, p53-IER5-HSF1 pathway, that contributes to stress resistance and tumor progression

Rieko Ohki 
National Cancer Center (Tokyo) 

The transcription factors p53 and Heat shock factor 1 (HSF1) both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both might have the potential to promote tumor development in a context-dependent manner. Here we show that a p53 target gene, Immediate early response gene 5 (IER5), encodes a novel activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. We also demonstrate that IER5 is a nuclear protein, and IER5 mutants harboring mutations at the nuclear localization motifs, and therefore can not be localized within the nucleus, can not generate the hypo-phosphorylated form of HSF1 nor activate HSF1. IER5 is transcriptionally upregulated in various cancers. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation and metastasis of these cells under various stressed conditions both in vitro and in vivo. Finally, high expression of IER5 and HSF1 target genes are both associated with poor prognosis of various cancer patients. These results reveal the existence of a novel p53/IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 and acquisition of stress resistance observed in various cancers.