Lung cancer is the number one cancer killer in the United States, exceeding breast, colorectal, prostate, and melanoma malignancies combined. Eighty percent of lung cancers are non-small cell lung cancers (NSCLC). Our laboratory has established that the transcription factor NF-KappaB is an important regulator of survival in NSCLC cells and we have found that NSCLC tumors display elevated NF-KB and IKK. This proposal will investigate the hypothesis that IKKalpha activation potentiates NF-kappaB transcription in a unique way; by phosphorylating and stimulating the nuclear export of the transcriptional co-repressor silencing mediator for retinoid and thyroid receptors (SMRT). SMRT plays a key role in regulating NF-KappaB-dependent transcription through its ability to recruit histone deacetylases, which are responsible for gene silencing. Data presented in this proposal indicate that IKKalpha translocates to the nucleus following stimulation. Chromatin-associated IKKalpha inversely correlates with SMRT and HDAC-associated complexes across endogenous NF-kappaB regulated promoters. IKKalpha phosphorylates SMRT in vitro and in vivo to stimulate nuclear export of this corepressor. However, to achieve full NF-KappaB transcriptional activity, IKKalpha must also phosphorylate RelA/p65 within its transactivation domain. Using cell reattachment assays we have shown that endogenous IKKot activity is required for SMRT nuclear export. Inhibition of this regulatory pathway inhibits NF-KappaB transcription and potentiates programmed cell death (anoikis). Experiments described in Aim 1 will establish whether IKKalpha is dysregulated in human NSCLC tumors and will identify the molecular signaling pathways responsible for inducing IKKalpha nuclear translocation. Aim 2 will identify the IKKalpha-induced phosphorylation sites within SMRT and determine the role of CRM-1 and 14-3-3 in nuclear export of SMRT. Additionally, experiments will determine if IKKalpha-mediated phosphorylation of RelA/p65 is responsible for liberating SMRT from NF-KappaB. Aim 3 will utilize xenograft mouse models to elucidate whether IKKalpha is required to maintain NSCLC tumor growth in nude mice. Moreover, transgenic mice, tissue-specifically expressing oncogenic K-Ras, will be utilized to determine the role of IKKalpha in primary lung cancer development. The overall goal of this proposal is to determine whether IKKalpha regulates NF-KappaB transcription by phosphorylating and inactivating the co-repressor SMRT. This effect would be predicted to have profound effects on transcription and the development of lung cancer. This new understanding would provide a useful NSCLC marker and would potentially identify an important molecular target that may result in novel treatment strategies for this deadly disease.