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    Jeanine Louise Frey Van Nostrand.
    The p53 protein plays a critical role in tumor suppression, as evidenced by its high mutation rate in human tumors and the observation that p53-null mice get cancer with 100% penetrance. p53 is a stress sensor that responds to cellular assaults, such as DNA damage or serum starvation, by inducing downstream effector functions, including apoptosis and cell cycle arrest. p53 itself is a transcription factor that promotes its downstream functions through its ability to transcriptionally activate downstream targets genes, although p53 also has transactivation-independent functions. However, the role that p53 plays in development and the role its target genes play in development and tumor suppression are not well defined. First, we sought to analyze the role of p53 transcriptional activation in development. Using knock-in mice expressing a p53 transactivation domain mutant (p5325,26,53,54), which does not bind Mdm2, we found that developmental defects caused by unrestrained p53 require p53's transactivation ability, as p5325,26,53,54/- mice are viable. Surprisingly, we also found that expression of p5325,26,53,54 in the presence of wild-type p53 induces embryonic lethality and a spectrum of phenotypes characteristic of a congenital syndrome known as CHARGE, including coloboma, inner and outer ear malformations, heart outflow tract defects, and craniofacial defects. We find that p5325,26,53,54 mutant protein is able to stabilize and hyperactivate wild-type p53, driving p53 to inappropriately induce target gene expression and trigger cell-cycle arrest and apoptosis during development. We further find that p53 is activated in CHD7-mutant CHARGE syndrome patient samples and upon genetic ablation of Chd7 in mouse cells. Additionally, p53-heterozygosity is able to partially rescue Chd7 null embryonic phenotypes, indicating that Chd7 deficiency provokes p53 activation and p53-dependent phenotypes. Thus, through the use of p5325,26,53,54 mice, we have uncovered a critical role for transactivation in provoking developmental defects induced by unrestrained p53 and revealed a novel and critical role for p53 in promoting human CHARGE syndrome. Subsequently, we sought to uncover the role of the p53 family as a whole, including the two related transcription factors p63 and p73, in development. While p53 null mice are viable, p63 null mice are perinatal lethal with epidermal and limb defects and p73 null mice typically die within the first two months of life and manifest neuronal defects. To determine if the absence of more severe developmental phenotypes is due to redundancy between the p53 family members, we bred and analyzed compound knockout mice. Analysis of compound knockout embryos revealed viability of all double knockout embryos (p53p63, p53p73, and p53p63) and five allele knockout embryos (double knockout and heterozygous for the remaining gene) during embryogenesis with the only defects detected being accounted for by loss of single p53 family members. Surprisingly, we also identified a single viable triple knockout at E10.5 with normal morphology except for hypoplastic cardiac cushions. Thus, these results suggest that p53 family members are neither critical nor redundant for relatively normal development. We next sought to determine the role of the p53 target gene Siva in both development and tumorigenesis. Our lab previously identified Siva as a p53 target gene critically important for p53-dependent apoptosis. Here we generated a Siva knockout mouse strain using gene-trap technology. Analysis of homozygous Siva mutant mice, revealed mid-gestational embryonic lethality associated with both embryonic and extra-embryonic defects, including developmental delay and defects of neural tube closure, yolk sac vasculature, and chorioallantoic fusion. The defects associated with Siva deficiency resembled those in embryos with loss of key components of TGF [beta]/SMAD signaling pathways, and notably, Siva null embryos displayed reduced SMAD protein levels and aberrant SMAD target gene expression. Thus, loss of Siva results in embryonic lethality associated with defects in SMAD signaling. Due to the embryonic lethality associated with Siva loss, we went on to generate a conditional Siva knockout mouse strain for studies of Siva in tumor suppression. Using a Kras-dependent non-small cell lung cancer model, we found surprisingly that Siva is necessary for efficient tumorigenesis in this model, as Siva-deficiency results in reduced tumor number and tumor burden. Additionally, Siva knockdown in non-small cell lung cancer cell lines resulted in reduced proliferation and tumorigenic potential. Furthermore, Siva loss inhibited mTOR signaling, induced autophagy, and reduced mitochondrial respiration, suggesting that Siva is necessary for normal metabolic function to promote proliferation. Moreover, Siva levels have prognostic ability for human non-small cell lung cancer patient survival. Therefore, our findings reveal that the p53 target gene Siva enables Kras-dependent lung cancer development and is necessary for oxidative phosphorylation, and that Siva levels could be used to estimate the prognosis of non-small cell lung cancer patient survival. Collectively, these results significantly expand our knowledge of the role of p53, the p53 family, and the p53 target gene Siva in development as well as the role of Siva in tumorigenesis. We found that unrestrained p53 results in developmental defects resembling CHARGE syndrome and that compound loss of the p53 family members does not promote profound developmental phenotypes other than those observed in single mutants. Moreover, we found that Siva is necessary for both proper embryonic development and efficient non-small cell lung cancer tumor development through roles in SMAD signaling and metabolism, respectively.
    Digital Access 2014
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