Tomoo Iwakuma

Gain of Function of a p53 Hot Spot Mutation in a Mouse Model of Li-Fraumeni Syndrome

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

The inherent instability of mutant p53 is alleviated by Mdm2 or p16INK4a loss

The p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. We generated mice with a missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance of these data, we examined the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation, and p16INK4a, a member of the Rb tumor suppressor pathway. Mice lacking Mdm2 or p16INK4a stabilized mutant p53, and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 will also stabilize mutant p53 with dire consequences.

Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system

Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5′ untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5′ splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 105–106 transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.

CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance

Emerging evidence indicates the presence of tumor-initiating cells (TIC) or cancer stem cells in osteosarcoma. However, no study has shown specific markers to identify osteosarcoma TICs with in vivo tumor formation ability. Additionally, there has been a lack of investigations gauging the contribution of osteosarcoma TICs to metastatic and drug-resistant properties. In this study, we have identified mouse and human osteosarcoma TICs using mesenchymal stem cell markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. Both mouse and human cells expressing these markers were sorted and analyzed for their abilities of tumor formation with as few as 200 cells, self-renewability, multipotency, drug resistance, metastatic potential, and enrichment of a metastasis-associated marker (CXCR4) and a drug resistance marker (ABCG2). CD117(+)Stro-1(+) cells efficiently formed serially transplantable tumors, whereas CD117(-)Stro-1(-) cells rarely initiated tumors. On orthotopic injections, CD117(+)Stro-1(+ )cell-derived tumors metastasized at a high frequency. Further, CD117(+)Stro-1(+) cells showed high invasive and drug-resistant properties and were efficiently enriched for CXCR4 (20-90%) and ABCG2 (60-90%). These results suggest possible mechanisms for the high metastatic and drug-resistant properties of osteosarcoma TICs. In summary, CD117 and Stro-1 identify osteosarcoma TICs associated with the most lethal characteristics of the disease-metastasis and drug resistance-and these markers offer candidates for TIC-targeted drug delivery aimed at eradicating osteosarcoma.

Increased Sensitivity to UV Radiation in Mice with a p53 Point Mutation at Ser389

ABSTRACT Phosphorylation is important for p53 protein stabilization and activation after DNA damage. Serine 389 of p53 is specifically phosphorylated after UV irradiation, whereas gamma radiation activates p53 through a different pathway. To study the in vivo significance of p53 phosphorylation at serine 389, we generated a physiological mouse model in which p53 phosphorylation at serine 389 is abolished by alanine substitution. Homozygous mutant p53.S389A mice are viable and have an apparently normal phenotype. However, cells isolated from these mice are partly compromised in transcriptional activation of p53 target genes and apoptosis after UV irradiation, whereas gamma radiation-induced responses are not affected. Moreover, p53.S389A mice show increased sensitivity to UV-induced skin tumor development, signifying the importance of serine 389 phosphorylation for the tumor-suppressive function of p53.

A High-Frequency Regulatory Polymorphism in the p53 Pathway Accelerates Tumor Development

MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2(SNP309T) or the MDM2(SNP309G) allele to address the impact of MDM2(SNP309G) on tumorigenesis. Mdm2(SNP309G/G) cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2(SNP309G/G) mice succumbed to tumors before 1 year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2(SNP309G) allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2(SNP309G) allele.

Heterodimerization of Mdm2 and Mdm4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability

Mdm2 and Mdm4 are homologous RING domain-containing proteins that negatively regulate the tumor suppressor p53 under physiological and stress conditions. The RING domain of Mdm2 encodes an E3-ubiquitin ligase that promotes p53 degradation. In addition, Mdm2 and Mdm4 interact through their respective RING domains. The in vivo significance of Mdm2-Mdm4 heterodimerization in regulation of p53 function is unknown. In this study, we generated an Mdm4 conditional allele lacking the RING domain to investigate its role in Mdm2 and p53 regulation. Our results demonstrate that homozygous deletion of the Mdm4 RING domain results in prenatal lethality. Mechanistically, Mdm2-Mdm4 heterodimerization is critical for inhibiting lethal p53 activation during early embryogenesis. However, Mdm2-Mdm4 interaction is dispensable for regulating p53 activity as well as the stability of Mdm2 and p53 at later stages of development. We propose that Mdm4 is a key cofactor of Mdm2 that inhibits p53 activity primarily during early embryogenesis but is dispensable for regulating p53 and Mdm2 stability in the adult mouse.

Ubiquitination of mammalian AP endonuclease (APE1) regulated by the p53–MDM2 signaling pathway

APE1/Ref-1 is an essential DNA repair/gene regulatory protein in mammals of which intracellular level significantly affects cellular sensitivity to genotoxicants. The apurinic/apyrimidinic endonuclease 1 (APE1) functions are altered by phosphorylation and acetylation. We here report that APE1 is also modified by ubiquitination. APE1 ubiquitination occurred specifically at Lys residues near the N-terminus, and was markedly enhanced by mouse double minute 2 (MDM2), the major intracellular p53 inhibitor. Moreover, DNA-damaging reagents and nutlin-3, an inhibitor of MDM2–p53 interaction, increased APE1 ubiquitination in the presence of p53. Downmodulation of MDM2 increased APE1 level, suggesting that MDM2-mediated ubiquitination can be a signal for APE1 degradation. In addition, unlike the wild-type APE1, ubiquitin–APE1 fusion proteins were predominantly present in the cytoplasm. Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells. These results reveal a novel regulation of APE1 through ubiquitination.

Targeting Oncogenic Mutant p53 for Cancer Therapy.

Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels.