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2005 Scientific Report

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Laboratory of Cancer and

Laboratory of Cancer and Developmental Cell Biology Nicholas S. Duesbery, Ph.D. Dr. Duesbery received both his M.Sc. (1990) and Ph.D. (1996) degrees in zoology from the University of Toronto, Canada, under the supervision of Yoshio Masui. Before his appointment as a Scientific Investigator at VARI in April 1999, he was a postdoctoral fellow in the laboratory of George Vande Woude in the Molecular Oncology Section of the Advanced BioScience Laboratories–Basic Research Program at the National Cancer Institute–Frederick Cancer Research and Development Center, Maryland. Staff Paul Spilotro, M.D. Philippe Depeille, Ph.D. Hilary Wagner, M.S. John Young, M.S. Elissa Boguslawski Laboratory Members Students Chia-Shia Lee Lisa Orcasitas Visiting Scientist Gustavo Nacheli, M.D. Research Interests The overall goal of the Laboratory of Cancer and Developmental Cell Biology is to further the study of mitogenactivated protein kinase kinase (MEK) signaling in health and disease. Currently, work performed in the lab is organized into three projects to explore 1) MEK signaling and tumor biology, 2) the therapeutic potential of anthrax lethal toxin (LeTx), and 3) molecular mechanisms of LeTx action. MEK signaling and tumor biology Many malignant sarcomas, such as angiosarcomas, are refractory to currently available treatments. However, sarcomas possess unique vascular properties which indicate they may be more responsive to therapeutic agents that target endothelial function. MEKs have been demonstrated to play an essential role in the growth and vascularization of carcinomas. We hypothesize that signaling through multiple MEK pathways is also essential for growth and vascularization of sarcomas. The objective of this research is to define the role of MEK signaling in the growth and vascularization of human sarcoma and to determine whether inhibition of multiple MEKs by agents such as anthrax lethal toxin, a proteolytic inhibitor of MEKs, may form the basis of a novel therapeutic approach to the treatment of human sarcoma. In 2004 we successfully obtained funding from the National Institutes of Health for this project. The therapeutic potential of anthrax lethal toxin Data from the National Cancer Institute’s Anti-Neoplastic Drug Screen indicates that several tumor types, notably melanomas and colorectal adenocarcinomas, are sensitive to LeTx. In addition, we have noted that angio-proliferative tumors are also very sensitive to LeTx treatment. Consequently we have undertaken a systematic evaluation of the effects of LeTx upon human tumor-derived melanomas, colorectal adenocarcinomas, and Kaposi’s sarcoma. The goal of this project is to develop novel therapeutic agents that may be efficacious in the treatment of human malignancies. In 2004 we successfully obtained funding from the National Institutes of Health to evaluate the therapeutic potential of LeTx in the treatment of Kaposi’s sarcoma. Molecular mechanisms of LeTx action The lethal effects of Bacillus anthracis have been attributed to an exotoxin that it produces. This exotoxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). EF is an adenylate cyclase and together with PA forms a toxin referred to as edema toxin. LF is a Zn 2+ -metalloprotease which together with PA forms a toxin referred to as lethal toxin. LeTx is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals. The goal of this project is to develop a detailed molecular understanding of 20

the LF/MEK interaction that will facilitate the development of therapeutic agents for anthrax. In 2004, we identified a cluster of surface-exposed residues of LF that are distal to the catalytic site and are essential for its catalytic activity (Fig. 1). Details of this study were published in the Journal of Biological Chemistry. Staff notes Sherrie Boone, who has served as a technician in the lab since 2001, has left us. She was replaced in February 2004 by John Young, an M.Sc. graduate from the University of Oregon. John has initiated studies of LeTx and Kaposi’s sarcoma and has played a significant role in our identification of novel regions of LF that are required for its activity. Xudong Liang, a postdoctoral fellow who joined us in 2002, has moved on to a new position at the University of Minnesota. In his place we welcome Paul Spilotro, who joined us in August. Paul is currently evaluating MEK signaling in human fibrosarcoma. Elissa Boguslawski and Lisa Orcasitas joined our team in September. Elissa will serve as our vivarium technician in charge of murine studies, including xenografts. Lisa is a Bridges to the Baccalaureate student and is currently making sarcoma tissue microarrays so that she can evaluate MEK signaling in human tumor samples. In the summer of 2004, the lab was joined by Mia Hemmes, an undergraduate student from Michigan State University, who undertook a cytogenetic analysis of a primary human sarcoma-derived cell line. As well, we hosted Ricky Gonzalez and Lynda Gladding, two Grand Rapids area high school students interested in careers in biological research. Ricky and Lynda evaluated the sensitivity of murine endothelial cells to LeTx. Figure 1. A surface plot of anthrax LF highlighting mutagenized residues. A space-filled surface plot of LF was generated using Protein Explorer freeware. Residues identified as being critical for LF activity are colored yellow (K294), green (L293), red (L514), purple (N516), and orange (R491). Residues found to play a neutral or marginal role in LF activity are white. The NH 2 -terminus of MEK is indicated in black. A magnified image of this region shows that the critical residues are organized side-by-side in a focused band (KLLNR), which lies at one end of the catalytic groove. 21

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