Role of IKK in NSCLC Modulation of SMRT and NF-kappab

IKK 在 NSCLC SMRT 和 NF-kappab 调节中的作用

• 批准号: 7218066
• 负责人: MARTY W MAYO
• 金额: $ 26.47万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-19 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7218066
• 关键词: Address; American; Anoikis; Apoptosis; Apoptotic; Biological Assay; Breast; Cancerous; Cell Death; Cell Nucleus; Cell Survival; Cells; Chromatin; Colorectal; Complex; Data; Development; Disease; Disruption; Event; Extracellular Matrix; Failure; Gene Silencing; Genetic Transcription; Goals; Growth; Histone H3; Histones; Human; Immunohistochemistry; In Vitro; Laboratories; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Molecular; Molecular Target; NF-kappa B; Newly Diagnosed; Non-Small-Cell Lung Carcinoma; Nuclear Export; Nuclear Translocation; Nude Mice; Numbers; Oncogenic; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Prostate; Recruitment Activity; Regulation; Regulatory Pathway; Repression; Role; Sampling; Signal Pathway; Signal Transduction; Silencing Mediator of Retinoid Thyroid Receptor; Structure of parenchyma of lung; Tissues; Transactivation; Transcriptional Activation; Transgenic Mice; Tumor Tissue; United States; Xenograft procedure; cancer cell; chromatin immunoprecipitation; in vivo; melanoma; mouse model; novel; p65; phosphatidylinositol 3,4,5-triphosphate; promoter; receptor; research study; transcription factor; tumor; tumor growth

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：肺癌是美国排名第一的癌症杀手，超过乳腺癌，结直肠癌，前列腺和黑色素瘤恶性肿瘤。 80％的肺癌是非小细胞肺癌（NSCLC）。我们的实验室已经确定，转录因子NF-kappab是NSCLC细胞中生存的重要调节剂，我们发现NSCLC肿瘤显示出升高的NF-KB和IKK。该提案将调查以下假设：ikkalpha激活以独特的方式增强了NF-kappab的转录。通过磷酸化和刺激视网膜类和甲状腺受体（SMRT）的转录共抑制沉默介质的核输出。 SMRT通过募集组蛋白脱乙酰基酶的能力来调节NF-kappab依赖性转录，这是负责基因沉默的关键作用。该提案中提出的数据表明，ikkalpha刺激后易位到核。与内源性NF-kappab调节的启动子跨染色质相关的Ikkalpha与SMRT和与HDAC相关的复合物成反比。 Ikkalpha在体外和体内磷酸化SMRT以刺激该核心压力的核输出。但是，为了实现全NF-kappab的转录活性，Ikkalpha还必须在其反式激活域内磷酸化rela/p65。使用细胞重新分析测定，我们已经表明，SMRT核输出需要内源性IKKOT活性。抑制这种调节途径可抑制NF-kappab转录并增强程序性细胞死亡（Anoikis）。 AIM 1中描述的实验将确定在人NSCLC肿瘤中Ikkalpha是否失调，并将确定负责诱导Ikkalpha核转运的分子信号传导途径。 AIM 2将确定SMRT内Ikkalpha诱导的磷酸化位点，并确定CRM-1和14-3-3在SMRT核出口中的作用。此外，实验将确定RELA/p65的Ikkalpha介导的磷酸化是否负责从NF-kappab释放SMRT。 AIM 3将利用异种移植小鼠模型来阐明是否需要Ikkalpha来维持裸鼠的NSCLC肿瘤生长。此外，将利用转基因小鼠，特定于表达致癌K-Ras的组织，以确定Ikkalpha在原发性肺癌发育中的作用。该提案的总体目标是确定Ikkalpha是否通过磷酸化和灭活共抑制剂SMRT来调节NF-kappab转录。预计这种影响会对转录和肺癌的发展产生深远影响。这种新的理解将提供有用的NSCLC标记，并有可能确定一个重要的分子靶标，该靶标可能会导致这种致命疾病的新治疗策略。