Somatic Trp53 mutations differentially drive breast cancer and evolution of metastases.

TP53 mutations are the most frequent genetic alterations in breast cancer and are associated with more aggressive disease and worse overall survival. We have created two conditional mutant Trp53 alleles in the mouse that allow expression of Trp53R172H or Trp53R245W missense mutations in single cells surrounded by a normal stroma and immune system. Mice with Trp53 mutations in a few breast epithelial cells develop breast cancers with high similarity to human breast cancer including triple negative. p53R245W tumors are the most aggressive and exhibit metastases to lung and liver. Development of p53R172H breast tumors with some metastases requires additional hits. Sequencing of primary tumors and metastases shows p53R245W drives a parallel evolutionary pattern of metastases. These in vivo models most closely simulate the genesis of human breast cancer and will thus be invaluable in testing novel therapeutic options.

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.

Multiple stress signals activate mutant p53 in vivo.

p53 levels are tightly regulated in normal cells, and thus, the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly murine double minute 2 (Mdm2), allowing p53 to be stabilized to activate cell-cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild-type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild-type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which oftentimes contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status.

Loss of Mdm4 results in p53-dependent dilated cardiomyopathy.

BACKGROUND: Although several loci for familial dilated cardiomyopathy (DCM) have been mapped, the origin of a large percentage of DCM remains unclear. Mdm2, a p53-negative regulator, protects cardiomyocytes from ischemic and reperfusion-induced cell death. Mdm4, a homolog of Mdm2, inhibits p53 activity in numerous cell types. It is unknown whether Mdm4 plays a role in the inhibition of p53 in fully differentiated tissues such as adult cardiomyocytes and whether this role is associated with DCM. METHODS AND RESULTS: The conditional knockout of Mdm4 in the heart by use of cardiomyocyte-specific Cre (alphaMyHC-Cre) allele does not result in any developmental defects. With time, however, mice with deletion of Mdm4 in the adult heart developed DCM and had a median survival of 234 days. More interestingly, the onset of DCM occurs significantly earlier in male mice than in female mice, which mimics human DCM disease. DCM in Mdm4 mutant mice was caused by loss of cardiomyocytes by apoptosis, and it was p53-dose dependent. CONCLUSION: Activity of p53 was inhibited by Mdm4 even in the fully differentiated cardiomyocyte. Elevated apoptosis mediated by the p53 pathway in cardiomyocytes may be a mechanism for DCM.

Synergistic roles of Mdm2 and Mdm4 for p53 inhibition in central nervous system development.

Loss of Mdm2 or Mdm4 leads to embryo lethal phenotypes that are p53-dependent. To determine whether Mdm2 and Mdm4 inhibit p53 function redundantly in a more restricted cell type, conditional alleles were crossed to a neuronal specific Cre transgene to delete Mdm2 and Mdm4 in the CNS. Mice lacking Mdm2 in the CNS developed hydranencephaly at embryonic day 12.5 due to apoptosis, whereas Mdm4 deletion showed a proencephaly phenotype at embryonic day 17.5 because of cell cycle arrest and apoptosis. The deletion of both genes, strikingly, contributed to an even earlier and more severe CNS phenotype. Additionally, Mdm2 and Mdm4 had a gene dosage effect, because loss of three of the four Mdm alleles also showed a more accelerated CNS phenotype than deletion of either gene alone. All phenotypes were rescued by deletion of p53. Thus, these in vivo data demonstrate the importance of Mdm4 independent of Mdm2 in inhibition of p53.

Tissue-specific differences of p53 inhibition by Mdm2 and Mdm4.

The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.