The NADPH-oxidase Nox1 can generate reactive oxygen that functions through various signaling pathways to activate mitogenic growth and angiogenesis. Nox1 is overexpressed in ~ 60-70% of early human colon cancers, suggesting a role in tumorigenesis. We find that introduction of mutationally activated K-Ras results in marked induction of Nox1 mRNA, and that K-Ras-induced cell growth is prevented by antioxidants and by siRNA to Nox1. We hypothesize that overexpression of Nox1 in human colon cancers can result from activation of Nox1 transcription by RasV12. Human tumors will be characterized to establish whether Nox1 overexpression is linked to oncogenic mutations in K-Ras. In addition, inactivating mutations in the tumor suppressors p53 and ARC and activation of MAP Kinase and PI 3-kinase pathways will be evaluated in these tumors. To evaluate the causal relationship between K-Ras oncogenic mutation, Nox1 overexpression, and cancer, Nox1 overexpressing and Nox1 knockout mice have been developed. A knock-in mouse expressing an inactive Nox1 fused to b-galactosidase will be used as a reporter of in vivo Nox1 promoter activity, and a Nox1 promoter-reporter construct will be used in a cellular system to define the Ras-related signal transduction systems downstream of K-Ras(V12) that regulate Nox1 mRNA expression. In addition, a minimal K-Ras regulated promoter will be defined using deletion and mutation analysis, Dnase hypersensitivity, and DNA gel shift and supershift approaches. These studies are expected to provide novel information regarding the role of Nox1 in gastrointestinal cancers, its relationship to oncogenic mutations in K-Ras and tumor suppressors, and the signaling systems utilized by Ras to regulate expression of Nox enzymes and levels of reactive oxygen species. ? ? ?
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