A number of important questions remain unanswered with respect to early events that limit tumor growth and progression as a neoplasm begins to become deprived of oxygen and nutrients. As this occurs cells experience hypoxia and acidosis that lead to activation of a p53-dependent checkpoint. Little is known about the molecular events upstream of p53 during hypoxia-induced checkpoint activation and what are the mediators of hypoxia-induced tumor suppression. Our working hypothesis is that hypoxia activates checkpoints that limit early tumor expansion and that this involves p53 and its transcriptional target genes. In addition there are multiple survival pathways selected for during hypoxia including p53 mutation, HIF activation, alteration in Myc signaling or BRCA1-dependent repair that may impact on tumor growth and survival. We propose to explore these pathways through the following specific aims:
Aim 1 : Investigate hypoxia-induced apoptotic checkpoint. A. Elucidate markers, kinases. Modifications that regulate p53 in vitro and in vivo;perform colocalization between p53 and its checkpoint regulators with regions of hypoxia using immunohistochemistry and immunofluorescence. B. Investigate apoptotic targets and their localization during hypoxia;utilize microarrays, chromatin immunoprecipitation and mass-spectrometry for analysis and to perform subsequent validation. C. Investigate p53 target genes in early damage checkpoint comparing normoxia with hypoxia in tumor samples.
Aim 2 : Examine requirements in tumor suppression of p53 targets and other pathways modulated by hypoxia in vivo. A. Utilize gene silencing, imaging, immunostaining and mouse xenografts to analyze the role of specific pro-apoptotic and growth inhibitory p53 target genes. B. Investigate cross-talk between p53, HIF, Myc and BRCA1 during hypoxia to unravel coordinated regulation in tumor suppression and deregulated function in cancer development. The proposed studies will provide insights into early events in tumor development with specific focus on hypoxia-induced checkpoints that must be suppressed. The work may lead to new strategies for limiting tumor growth at its earliest stages.
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