Activation of NF-kB by DNA-damaging anticancer agents, including ionizing radiation (IR), has emerged as an important modulator of malignant cell behaviors, such as resistance to apoptotic cell death. This signal transduction pathway also serves as a paradigm to understand how nuclear DNA damage may induce nucleus-to-cytoplasmic signal transduction pathways. We have previously discovered a novel nucleus-to-cytoplasmic NF-kB signaling pathway induced by IR and other agents that can induce DNA double strand breaks (DSBs). This signaling pathway involves a post-translational modification (PTM) of NEMO (NF-kB essential modulator)/IKK3, the regulatory subunit of the I:B kinase (IKK) complex, by SUMO-1 (small ubiquitin-like modifier 1). We now generated a novel NemoDK knockin mice harboring a germ-line mutation of the sumoylation sites. In the current proposal, we will establish the physiological importance of this new NF-kB signaling pathway in response to IR by directly evaluating the role of NEMO sumoylation in vivo and further dissect the critical upstream and downstream mechanisms. The following three aims will address our central hypothesis that NEMO sumoylation plays a critical physiological role in mediating NF-kB activation by IR to modulate radiation sensitivity in vivo:
Aim 1 : Determine the roles of NEMO sumoylation in modulating radiation sensitivity in vivo.
Aim 2 : Reveal the upstream role of NEMO zinc finger in promoting NEMO sumoylation.
Aim 3. Elucidate SUMO-1 specific downstream regulation of NEMO function. We believe that the proposed research is important for two major reasons. First, the described research will uncover novel NF-kB signal transduction mechanisms in response to DNA damage stimuli. Second, understanding the mechanisms of NF-kB activation by DNA damaging agents may help identify novel drug targets to improve the current methods of cancer therapy.

Public Health Relevance

Many anticancer agents, including ionizing radiation (IR), are used to kill cancer cells, but unfortunately they also turn on cancer cell survival mechanisms in cancer cells that counter the death effect. One of these mechanisms is the activation of the transcription factor NF-kB that rapidly turns on the synthesis of survival genes. The proposed research will uncover important mechanisms involved in this activation, which will help identify novel drug targets to improve the current methods of cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA077474-15
Application #
8633938
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Arya, Suresh
Project Start
2000-06-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
15
Fiscal Year
2014
Total Cost
$269,746
Indirect Cost
$85,965
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Jackson, Shawn S; Miyamoto, Shigeki (2015) Dissecting NF-κB signaling induced by genotoxic agents via genetic complementation of NEMO-deficient 1.3E2 cells. Methods Mol Biol 1280:197-215
Jackson, Shawn S; Oberley, Christopher; Hooper, Christopher P et al. (2015) Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling. Exp Cell Res 331:58-72
Hwang, Byounghoon; McCool, Kevin; Wan, Jun et al. (2015) IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation. J Biol Chem 290:17967-84
Hwang, Byounghoon; Phan, Funita P; McCool, Kevin et al. (2015) Quantification of cellular NEMO content and its impact on NF-κB activation by genotoxic stress. PLoS One 10:e0116374
Shin, Eun Myoung; Hay, Hui Sin; Lee, Moon Hee et al. (2014) DEAD-box helicase DP103 defines metastatic potential of human breast cancers. J Clin Invest 124:3807-24
Hooper, Christopher; Jackson, Shawn S; Coughlin, Emma E et al. (2014) Covalent modification of the NF-κB essential modulator (NEMO) by a chemical compound can regulate its ubiquitin binding properties in vitro. J Biol Chem 289:33161-74
Berry, Scott M; Chin, Emily N; Jackson, Shawn S et al. (2014) Weak protein-protein interactions revealed by immiscible filtration assisted by surface tension. Anal Biochem 447:133-40
Jackson, Shawn S; Coughlin, Emma E; Coon, Joshua J et al. (2013) Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification. Protein Expr Purif 92:48-53
Pak, Chorom; Miyamoto, Shigeki (2013) A new alpha in line between KRAS and NF-κB activation? Cancer Discov 3:613-5

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