INTRAGENIC SUPPRESSOR MUTATIONS FOR P53 CANCER MUTATIONS

Washington University, Saint Louis MO

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Abstract

The p53 protein exerts its tumor suppressor function mainly as a transcription factor that induces GI and G2 cell cycle arrest and/or apoptosis. Approximately 50 percent of human cancers have p53 missense mutations that result in high levels of p53 cancer mutants with a structurally altered core domain unable to bind to P53 DNA-binding sites upstream of effector genes. The same cancers (corresponding to 2.4 million cancer patients per year worldwide) carry a poor prognosis since p53 is an important mediator of cancer therapy-induced apoptosis. If one could restore function to this large pool of p53 mutant proteins, the impact on cancer therapy would be substantial. A detailed knowledge is therefore needed of the extent to which the most common p53 cancer mutations destabilize the structure of the p53 core domain. More importantly, suppressor mechanisms must be identified that can override the deleterious effects of p53 cancer mutations. This goal can be achieved through the study of intragenic suppressor mutations. A genetic approach using our functional p53 yeast assay showed in a pilot study that such suppressor mutations exist for selected p53 cancer mutations and that they can be isolated very efficiently. We now propose to identify intragenic suppressor mutations for the eight most common p53 cancer mutations and test them against the 35 most common p53 cancer mutations (accounting for 37 percent of human cancers with p53 mutations) using yeast and mammalian assays. This systematic study will define the structural motifs that are absolutely essential for the structural integrity of the p53 core domain and may identify suppressor mutations that act as global suppressors. The crystal structures for the core domains of the most promising suppressor mutations, the most common cancer mutations and combinations of both will then be determined to acquire a precise understanding of the structural dynamics of the p53 core domain. Preliminary experiments indicate that p53 proteins with a suppressor mutation alone have characteristics different from wild-type p53. Some of them may, in fact, behave like "super p53" proteins that are resistant to the dominant-negative effects of p53 cancer mutants and that may be more efficient in inducing cell cycle arrest and/or apoptosis. We will identify such p53 proteins and determine the mechanisms that make them resistant to dominant-negative p53 cancer mutants. We will further study how some of them are able to induce selectively and/or strongly G1 or G2 arrest or apoptosis. This will determine the relative importance of p53 downstream effector genes and may ultimately lead to the identification of new p53 pathways.