TRAIL, a ligand for death receptors (DRs), is considered a potential anti-cancer agent, as it shows selective high cytotoxicity toward tumor cells and little or no toxicity against normal cells. Currently, a recombinant TRAIL and agonistic antibodies directed at DRs are in phase-II clinical trials. However, recent studies have demonstrated that many types of cancer cells possess intrinsic or acquired resistance to TRAIL. Moreover, TRAIL application has been found to activate NF-kB and enhance metastasis in apoptosis-resistant cancer cells. Gene knockout studies have demonstrated that caspase-8 activity is essential not only for TRAIL-induced cell death, but also for TRAIL- induced NF-kB activation. At present, it is believed that fully activated caspase-8 induces apoptosis whereas partially activated caspase-8 activates NF-kB. However, the caspase-8 substrates that mediate this form of NF-kB activation have not been identified. We have identified RIP1 as a caspase-8 substrate that mediates TRAIL- induced NF-kB activation, discovered that caspase-8 cleaves RIP1 at three sites, and found that this cleavage is regulated in vivo by cFLIP. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 at all three sites in response to TRAIL treatment, resulting in rapid RIP1 depletion and the induction of apoptosis;in apoptosis-resistant cells, however, TRAIL induces RIP1 cleavage mainly at one site, producing a constitutively active form of RIP1 (p60RIP1n) that activates the NF-kB pathway. Notably, overexpression of cFLIP is sufficient to trigger limited RIP1 cleavage and the accumulation of p60RIP1n. Importantly, in Hodgkin's lymphoma, cFLIP is overexpressed and a portion of RIP1 is constitutively processed to p60RIP1n. These data suggest that cFLIP-regulated, caspase- 8-mediated limited cleavage of RIP1 promotes NF-kB activation, and that such cleavage occurs constitutively in certain human cancers. These findings support our central hypothesis that cFLIP overexpression restricts TRAIL- induced caspase-8 activation to a moderate level, promoting RIP1 processing to p60RIP1n and, thereby, NF-kB activation. The objective of the proposed study is to evaluate the influence of cFLIP on caspase-8-mediated RIP1 cleavage, dissect the mechanisms by which RIP1 cleavage modulates NF-kB activation in response to TRAIL stimulation, and determine the pathological role of RIP1 cleavage in cancer cell resistance to TRAIL-induced apoptosis. To achieve these objectives, we propose to carry out the following specific aims: 1) determine the role of caspase-8-mediated RIP1 cleavage in promoting TRAIL-induced NF-kB activation versus cell death;2) characterize the mechanisms by which caspase-8-mediated RIP1 cleavage activates NF-kB and inhibits cell death;3) assess the pathophysiological relevance of RIP1 cleavage in cancer cell resistance to TRAIL-induced apoptosis. The proposed work will define the mechanisms that underlie TRAIL-induced NF-kB activation, and guide the development of strategies to maximize the effectiveness of TRAIL as an anti-cancer agent.

Public Health Relevance

TRAIL, a potential anti-cancer agent, has been found to enhance metastasis in TRAIL-resistant cancer cells by activating NF-kB in a caspase-8-dependent manner. We identified caspase-8 substrate that mediates this form of NF-kB activation. Our work will not only shed new light on the mechanism by which TRAIL activates NF- kB, but will also provide rationale for maximizing the potential effectiveness of TRAIL as an anti-cancer agent.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA138475-03
Application #
8206767
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Arya, Suresh
Project Start
2010-02-23
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
3
Fiscal Year
2012
Total Cost
$301,913
Indirect Cost
$100,638
Name
University of Iowa
Department
Pathology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Zhang, Laiqun; Blackwell, Ken; Workman, Lauren M et al. (2016) TRAF2 exerts opposing effects on basal and TNF?-induced activation of the classic IKK complex in hematopoietic cells in mice. J Cell Sci 129:1455-67
Zhang, Laiqun; Blackwell, Ken; Workman, Lauren M et al. (2015) RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-?B Activation and Lymphoma Survival. Mol Cell Biol 35:3324-38
Zhang, Laiqun; Dittmer, Martin R; Blackwell, Ken et al. (2015) TRAIL activates JNK and NF-?B through RIP1-dependent and -independent pathways. Cell Signal 27:306-14
Blackwell, Ken; Zhang, Laiqun; Workman, Lauren M et al. (2013) Two coordinated mechanisms underlie tumor necrosis factor alpha-induced immediate and delayed I?B kinase activation. Mol Cell Biol 33:1901-15
Workman, Lauren M; Habelhah, Hasem (2013) TNFR1 signaling kinetics: spatiotemporal control of three phases of IKK activation by posttranslational modification. Cell Signal 25:1654-64
Shen, Rhine R; Zhou, Alicia Y; Kim, Eejung et al. (2012) I?B kinase ? phosphorylates TRAF2 to promote mammary epithelial cell transformation. Mol Cell Biol 32:4756-68
Zhang, Laiqun; Blackwell, Ken; Altaeva, Aliya et al. (2011) TRAF2 phosphorylation promotes NF-?B-dependent gene expression and inhibits oxidative stress-induced cell death. Mol Biol Cell 22:128-40
Habelhah, Hasem (2010) Emerging complexity of protein ubiquitination in the NF-?B pathway. Genes Cancer 1:735-747