We have used genetic methods to isolate mutant cell lines in which the activation of NFKappaB is constitutive. Complementation of these cells by overexpressing proteins that suppress the constitutive activation of NFKappaB has led to the functional identification of several novel candidate negative regulators. Constitutive activation of NFKappaB is common in many different cancers, where the ability of NFKappaB-regulated gene products to suppress apoptosis is important. We will investigate how different negative regulators control pathways that can lead to the activation of NFKappaB in response to many different cellular stresses by pursuing three specific aims:
Specific Aim 1 A: Validation of candidate negative regulators of NFKB and identification of new candidates. We will ablate the expression of candidate negative regulators in normal cells and test for consequent constitutive activation of NFKB. We will also test the effect of each candidate negative regulator in constitutive mutant cells that represent different complementation groups and that have biochemically distinct phenotypes. Also, we will test whether these proteins affect the induction of NFKappaB in response to IL-1 and TNF. To identify additional candidate negative regulators, we will complement additional constitutive mutants by using retroviral cDNA libraries and also by using Random Activation of Gene Expression, a novel technology that can drive high expression of any endogenous gene.
Specific Aim 1 B: Negative regulators of NFKappaB and cancer. We will determine the levels of expression of the novel negative regulators already in hand, and of additional ones to be isolated, in a variety of cancers in which NFKappaB is constitutively active, to identity tumor types in which loss of a particular regulator is frequent. We will then begin to explore the potential of using negative regulators as diagnostic aids and as possible targets for therapy.
Specific Aim 2 : Analysis of each validated negative regulator. A variety of assays will be employed to ascertain the physiological role of each validated negative regulator. We will analyze their effects on IKappaBalpha phosphorylation and on the activation of kinases already known to be involved in constitutive or induced NFKappaB activation. We will identify proteins associated with negative regulators by using co-immunoprecipitations, GST pull-downs and yeast two-hybrid analyses. We will also perform detailed structure-function analyses of negative regulators to help elucidate their mechanisms of action.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA095851-05
Application #
7238496
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Yassin, Rihab R,
Project Start
2003-06-13
Project End
2008-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
5
Fiscal Year
2007
Total Cost
$258,227
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Stark, George R; Darnell Jr, James E (2012) The JAK-STAT pathway at twenty. Immunity 36:503-14
Sears, Nathaniel; Sen, Ganes C; Stark, George R et al. (2011) Caspase-8-mediated cleavage inhibits IRF-3 protein by facilitating its proteasome-mediated degradation. J Biol Chem 286:33037-44
Wan, Youzhong; Kim, Tae Whan; Yu, Minjia et al. (2011) The dual functions of IL-1 receptor-associated kinase 2 in TLR9-mediated IFN and proinflammatory cytokine production. J Immunol 186:3006-14
Guo, Canhui; Stark, George R (2011) FER tyrosine kinase (FER) overexpression mediates resistance to quinacrine through EGF-dependent activation of NF-kappaB. Proc Natl Acad Sci U S A 108:7968-73
Lu, Tao; Jackson, Mark W; Wang, Benlian et al. (2010) Regulation of NF-kappaB by NSD1/FBXL11-dependent reversible lysine methylation of p65. Proc Natl Acad Sci U S A 107:46-51
Lu, Tao; Jackson, Mark W; Singhi, Aatur D et al. (2009) Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFkappaB. Proc Natl Acad Sci U S A 106:16339-44
Dasgupta, Maupali; Agarwal, Mukesh K; Varley, Patrick et al. (2008) Transposon-based mutagenesis identifies short RIP1 as an activator of NFkappaB. Cell Cycle 7:2249-56
Kandel, Eugene S; Lu, Tao; Wan, Youzhong et al. (2005) Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB. Proc Natl Acad Sci U S A 102:6425-30
Lu, Tao; Stark, George R (2004) Cytokine overexpression and constitutive NFkappaB in cancer. Cell Cycle 3:1114-7