Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis.

Characterization of cancer-associated missense mutations in MDM2.

MDM2 is an E3 ubiquitin ligase that binds the N-terminus of p53 and promotes its ubiquitin-dependent degradation. Elevated levels of MDM2 due to overexpression or gene amplification can contribute to tumor development by suppressing p53 activity. Since MDM2 is an oncogene, we explored the possibility that other genetic lesions, namely missense mutations, might alter its activities. We selected mutations in MDM2 that reside in one of the 4 key regions of the protein: p53 binding domain, acidic domain, zinc finger domain, and the RING domain. Unexpectedly, we observed that individual mutations in several of these domains compromised the ability of MDM2 to degrade p53. Mutations in the N-terminal p53 binding domain prevented the formation of a p53-MDM2 complex, thereby protecting p53 from degradation. Additionally, as would be predicted, several cancer-associated mutations in the RING finger domain disrupted the ubiquitin ligase activity of MDM2 and prevented p53 degradation. Interestingly, we observed that amino acid substitutions at the same codon differentially affected MDM2 activity. Our data reveal that mutations in this oncogene can have the paradoxical effect of suppressing its activity. Further understanding of how these mutations perturb MDM2 function may yield novel approaches to inhibiting its activity.

Mutant p53 protects ETS2 from non-canonical COP1/DET1 dependent degradation.

Mutations in the tumor suppressor gene TP53 contribute to the development of approximately half of all human cancers. One mechanism by which mutant p53 (mtp53) acts is through interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with the erythroblastosis virus E26 oncogene homologue 2 (ETS2), an ETS transcription factor, and increases its protein stability. To study the mechanism underlying ETS2 degradation, we knocked down ubiquitin ligases known to interact with ETS2. We observed that knockdown of the constitutive photomorphogenesis protein 1 (COP1) and its binding partner De-etiolated 1 (DET1) significantly increased ETS2 stability, and conversely, their ectopic expression led to increased ETS2 ubiquitination and degradation. Surprisingly, we observed that DET1 binds to ETS2 independently of COP1, and we demonstrated that mutation of multiple sites required for ETS2 degradation abrogated the interaction between DET1 and ETS2. Furthermore, we demonstrate that mtp53 prevents the COP1/DET1 complex from ubiquitinating ETS2 and thereby marking it for destruction. Mechanistically, we show that mtp53 destabilizes DET1 and also disrupts the DET1/ETS2 complex thereby preventing ETS2 degradation. Our study reveals a hitherto unknown function in which DET1 mediates the interaction with the substrates of its cognate ubiquitin ligase complex and provides an explanation for the ability of mtp53 to protect ETS2.

Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities.

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Synthesis of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uridines and study of their cytotoxic activity.

A series of uridine analogues modified at the 5-position with the 5-[alkoxy-(4-nitrophenyl)-methyl] moiety was synthesized. Nucleosides were formed as a mixture of two diastereoisomers, which were separated and tested for their cytotoxic activity in vitro against different cancer cell lines and for antimicrobial activity. Relationships between structure and the above mentioned activities were studied. The cytotoxic activity was slightly increased in some cases by transformation of bases to nucleosides. Depending on the length of the alkyl chain increased cytotoxic and antimicrobial activity were noted. The cytotoxic activity of the nucleosides was not due to cell cycle alterations, DNA and/or RNA synthesis.