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Dissecting signaling pathways to test cancer-killing agents

Institution: University of California, San Diego
Investigator(s): Yi Chen, Ph.D.
Award Cycle: 1998 (Cycle 7) Grant #: 7FT-0150 Award: $69,694
Subject Area: Cancer
Award Type: Postdoctoral Fellowship Awards
Abstracts

Initial Award Abstract
A variety of anticancer agents have been noted to be less effective in killing tumor cells than otherwise expected. Recently, this reduced effectiveness has been suggested to result from the anticancer agent-induced activation of transcription factors of the NF-B family, which form key components of a primary signal transduction pathway (i.e., a cellular messaging system). Activation of transcription factor NF-B has been shown to prevent programmed cell death, a biological pathway by which many anticancer agents kill tumor cells. Therefore, inhibiting NF-B activity may increase the effectiveness of certain cancer-killing agents. When cells are experimentally treated with NF-B activators, the activation process is initiated via a signaling cascade(s) involving several sequential components of NF-B. Currently, the steps leading to NF-B activation remain unknown and most of the components of this pathway have not been identified. One of strategies of killing tumor cells is to design therapeutic agents to block these signaling pathways by targeting one or more of their components. In this project, we are going to characterize some of the components mediating the activation of NF-B. These findings may be utilized to design therapeutic agents to block activation of NF-B, therefore improving the effectiveness of cancer-killing agents.

Final Report
Tumor cells can be killed by drugs through programmed cell death (PCD) called apoptosis. Many of these drugs, however, can also activate mechanism that prevents cells from apoptosis. Therefore, those drugs are less effective in killing tumor cells than otherwise expected. Recently, this reduced effectiveness has been suggested to result from the anticancer agent-induced activation of transcription factor of the NF-B family, which form key components of a primary signal transduction pathway. The NF-B activity is controlled by a kinase complex, called IB kinase (IKK). IKK contains at least three IKK polypeptides. Two of these polypeptides, IKKand IKK, are serine-threonine kinases. The third polypeptide, IKK, is a regulatory factor. The aims of this project are: 1) to study in vitro structure and function relationship among IKK subunits; 2) to study the mechanism by which IKK and IKK are regulated; 3) to characterize upstream signaling pathways to activate IKK; 4) to study the mechanism by which IKK is inhibited by some compounds.

Using baculovirus expression system, I was able to purify IKK three polypeptides to homogeneity. I have demonstrated that both IKK and IKK are direct kinases of IB proteins which regulate NF-B activation. Results from in vitro reconstitution experiments showed that IKK and IKK could independently phosphorylate their substrates although they form heterodimer in vivo. Binding to IKK enhances activities of both kinases. Previous data showed that homo- or hetero-dimerization of IKK and IKK is necessary for IKK activity and is mediated by the sequence located in the middle region of IKK and IKK. My experiments further demonstrated that C-terminal sequences of IKK and IKK are also required for dimerization. This explains previous observation that kinases were inactive when C-terminal sequences were deleted. Collaboration with other researchers, we have identified some signaling pathways to IKK activation. Based on above studies, we were able to study the mechanism by which IKK were inhibited by some compounds that inhibited NF-B activation. The most important observation made from IKK activation inhibition experiments is that many compounds inhibit IKK or IKK strongly but they do not inhibit IKK complex or IKK-associated IKK or IKK.

Characterization of IKK may provide important information for designing or screening agents that can reduce IKK activity. Using purified recombinant wild type or mutated IKK molecules, we have demonstrated that anti-inflammatory cyclopentenons prostaglandins can directly inhibit IKK and IKK activation. Studies from other laboratory also showed that another anti-inflammatory aspirin binds and inhibits IKK activation. However, in most cell types, majority of IKK and IKK are associate with IKK. Therefore, characterization of IKK complex is the most important next step needed to do and results from this study should be directly applicable to drug-designing or searching program and will help cancer-killing agents to treat many kinds of cancers, including lung cancer.
Publications

Positive and negative regulation of I kappa B kinase activity through IKK beta subunit phosphorylation
Periodical: Science Index Medicus:
Authors: Delhase M, Hayakawa M, Chen Y, Karin M ART
Yr: 1999 Vol: 284 Nbr: Abs: Pg: 309-313

Inhibition of NF- (kappa) B activation by arsenite through reaction with a critical cysteine in the activation loop of I (kappa) B Kinase.
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Kapahi P, Takahashi T, Natoli G, Adams SR, Chen Y, Tsien RY, and Karin M ART
Yr: 2000 Vol: Nbr: Abs: Pg:

NAK is an ikappaB kinase-activating kinase.
Periodical: Nature Index Medicus:
Authors: Tojima Y, Fujimoto A, Delhase M, Chen Y, Hatakeyama S, et al ART
Yr: 2000 Vol: 404 Nbr: 6779 Abs: Pg: 778-782

Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of ikappaB kinase.
Periodical: Nature Index Medicus:
Authors: Rossi A, Kapahi P, Natoli G, Takahashi T, Chen Y, Karin M, and Santoro MG ART
Yr: 2000 Vol: 403 Nbr: 6765 Abs: Pg: 103-108

JNK2 and IKKbeta are required for activating the innate response to viral infection.
Periodical: Immunity Index Medicus:
Authors: Chu WM, Ostertag D, Li ZW, Chang L, Chen Y, Hu Y, Williams B, Perrault J, and Karin M ART
Yr: 1999 Vol: 11 Nbr: 6 Abs: Pg: 721-731