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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Pathology B Study Section (PTHB)
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Spalholz, Barbara A
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University of Virginia
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