Elizabeth V. Wattenberg - US grants

DESCRIPTION: The mechanisms by which tumor promoters contribute to carcinogenesis remain unclear. Palytoxin presents a unique tool for investigating molecular mechanisms underlying tumor promotion. This novel tumor promoter is as potent as the phorbol esters in the two-stage mouse skin model, but does not activate protein kinase C. Investigating the biochemical mechanisms of action of palytoxin may help reveal critical targets in tumor promotion. Preliminary studies show that palytoxin activates c-Jun N-terminal kinase (JNK) in Swiss 3T3 fibroblasts. JNK may be a significant mediator of palytoxin action. JNK phosphorylates the proto-oncogene product c-Jun, which is a transcription factor, on Ser-63 and Ser-73 in vitro and in whole cells. Phosphorylation of c-Jun on Ser-63 and Ser-73 results in transcriptional activation. This observation suggests that palytoxin may modulate gene expression by activating JNK, which in turn phosphorylates and activates c-Jun. Phorbol esters can modulate c-Jun activity through a different signaling pathway. Therefore, modulation of c-Jun function may play a significant role in tumor promotion. The focus of this study is to investigate the hypothesis that palytoxin modulates c-Jun function through the regulation of JNK. The specific aims for this study are: Specific Aim 1. Characterize the activation of JNK by palytoxin in Cos7 cells. These studies will extend the model for investigating the regulation of JNK by palytoxin to a transfectable cell line to be used in Specific Aims 2 and 3. Specific Aim 2. Elucidate the signaling cascade that regulates activation of JNK by palytoxin. These studies will determine if palytoxin regulates JNK through activation of all or part of a protein kinase cascade that has recently been shown to regulate JNK activation (the Rac, Cdc42/Pak1/MEKK1/SEK1/JNK pathway). Specific Aim 3. Determine if palytoxin stimulates c-Jun transcriptional activity via JNK. c-Jun activation will be measured using a luciferase reporter gene system that has been used by others to demonstrate the regulation of c-Jun by other JNK activators. The results from these studies will determine if c-Jun is a common target for diverse skin tumor promoters, which do or do not function through protein kinase C. These studies may reveal a biochemical mechanism for the transmission of palytoxin-induced signals, and may also suggest a role for JNK in carcinogenesis.

[unreadable] DESCRIPTION (provided by applicant): The focus of this proposal is to investigate the critical biochemical characteristics of cells that express the oncogene Ras which make them susceptible to stimuli and events that advance the process of carcinogenesis. The proposed research will investigate the novel concept that phosphatases involved in a negative feedback loop that constrains Ras-stimulated signaling are vulnerable targets during early stages in carcinogenesis. We believe that carcinogenic agents disrupt the normal mechanisms by which cells compensate for the presence of activated Ras, and thus unleash super-activated signaling cascades. The role of aberrant regulation of signaling in carcinogenesis has been primarily studied by direct stimulation of signaling pathways. The proposed studies are innovative because they focus on an important alternative mechanism by which signaling pathways can become disregulated. Specifically, the proposed studies are designed to investigate the hypothesis that at early stages in carcinogenesis the activity of Ras-stimulated signaling pathways is inhibited by the upregulation of phosphatases. The subsequent down-regulation of the phosphatases releases Ras-stimulated pathways from negative constraints, which in turn results in aberrant activation of pathways that control cell fate and function. Activation of Ras is a frequent early event in human cancers, including lung and colon cancers, which are two of the most common cancers in the U.S. Thus, there is an urgent need to understand how the presence of activated Ras oncogenes sensitizes cells to agents and events that advance carcinogenesis. Specific Aims 1 and 2 are designed to use a variety of biochemical and molecular techniques to elucidate the mechanisms by which phosphatases that are important negative regulators of Ras-stimulated pathways are modulated in cells that express activated Ras. Specific Aim 3 is designed to investigate the functional consequences of the disregulation of such phosphatases in cells that express activated Ras. The recognition that aberrant regulation of phosphatases is associated with various disorders, including diabetes, obesity, and cancer, has sparked an interest in phosphatases as potentially important therapeutic targets. We expect the proposed studies to provide fundamental information on how disrupting the regulation of phosphatases can contribute to the process of carcinogenesis. Such insight will help in the identification of new diagnostic tools, the development of new strategies to block the advancement of carcinogenesis, and the development of chemotherapeutic agents to treat cancers that are characterized by aberrant Ras activity. [unreadable] [unreadable]