This proposal focuses on studying the role of 14-3-3? in regulating the MDM2-MDMX-p53 feedback pathway in response to cellular stress caused by hypoxia. Hypoxia plays a crucial role in tumorigenesis and particularly in the progression of solid tumors, as all solid tumors display 0%-8% oxygen concentrations in their hypoxic regions due to insufficient blood supply in rapidly growing tumors. During this pathological period, the tumor suppressor p53 is induced and activated to prevent the growth of the tumors by inducing apoptosis and cell growth arrest as well as slowing down angiogenesis. Although the role of p53 in suppressing tumor progression in response to hypoxia has been well established, the precise mechanism underlying hypoxia-triggered induction of p53 remains poorly understood. Biochemical and genetic studies have demonstrated that the ubiquitin-mediated proteasomal turnover and activity of the p53 tumor suppressor are tightly controlled by two oncoproteins called MDM2 and MDMX. Both of the proteins work together as p53 negative feedback regulators, because MDM2 is also a transcriptional target for p53 and MDMX acts as an MDM2 partner. Thus, untying this feedback loop is essential for activating p53 in order to prevent cells from undergoing transformation and neoplasia in response to various stresses. However, it is still unclear whether hypoxia induces p53 by affecting this loop. Clues from our recent studies suggest that 14-3-3? may play a role in the hypoxia-p53 pathway. In light of our recent work as well as studies by others, I hypothesize that hypoxia may induce the association of 14-3-3? with MDMX that is phosphorylated by the ATR-ChK1 cascade, consequently suppressing MDMX activity and leading to p53 activation. Therefore, this proposal will examine this hypothesis by addressing three specific aims: 1. To determine if ATR and ChK1 play a role in regulating the 14-3-3?-MDMX interaction in response to hypoxia;2. To elucidate the mechanisms underlying hypoxia-induced 14-3-3?-MDMX interaction and p53 activation;3. To determine if loss of 14-3-3? alleviates p53 response to hypoxia in cell culture and in animals. Successful implementation of these proposed studies will not only elucidate a novel mechanism by which p53 is induced in response to hypoxia, but would also identify 14-3-3? as another important player in cell growth control and tumorigenesis.
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