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2002 Scientific Report

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  • Michigan
  • Molecular
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Laboratory of Molecular

Laboratory of Molecular Oncology George F. Vande Woude, Ph.D. Dr. Vande Woude received his M.S. (1962) and Ph.D. (1964) from Rutgers University. From 1964–1972, he served first as a postdoctoral research associate, then as a research virologist for the U.S. Department of Agriculture at Plum Island Animal Disease Center. In 1972, he joined the National Cancer Institute as Head of the Human Tumor Studies and Virus Tumor Biochemistry sections and, in 1980, was appointed Chief of the Laboratory of Molecular Oncology. In 1983, he became Director of the Advanced Bioscience Laboratories-Basic Research Program at the National Cancer Institute’s Frederick Cancer Research and Development Center, a position he held until 1998. From 1995, Dr. Vande Woude first served as Special Advisor to the Director, and then as Director for the Division of Basic Sciences at the National Cancer Institute. In 1999, he was recruited to the Directorship of the Van Andel Research Institute in Grand Rapids, Michigan. Staff Rick Hay, M.D., Ph.D. Chong-Feng Gao, Ph.D. Chong-Chou Lee, Ph.D. Ling-Mei Wang, Ph.D. Yu-Wen Zhang, Ph.D. Dafna Kaufman, M.S. Meg Gustafson, B.A. Yanli Su, A.M.A.T. Mary Beth Bruch Laboratory Members Visiting Scientists & Staff Nariyoshi Shinomiya, M.D., Ph.D. Galia Tsarfaty, M.D. Ilan Tsarfaty, Ph.D. Students Daphna Atias Marketta Hassen Yasser Jimenez Jason Johnson Nathan Lanning Adi Laser, B.S. Kofi Obeng Dan Wohns Research Projects Research conducted in the Laboratory of Molecular Oncology uses a broad range of approaches to elucidate the molecular basis of cancer and to develop new agents for the diagnosis and therapy of cancer. We are primarily interested in the expression and activities of the receptor tyrosine kinase known as Met, its interactions with the ligand hepatocyte growth factor/scatter factor (HGF/SF), and the intracellular events influenced by Met activation. Aberrant expression of this receptor–ligand pair confers an invasive/metastatic phenotype in model systems of cancer. Inappropriate HGF/SF-Met expression occurs in most types of human solid tumors and is associated with poor clinical prognosis. Biochemistry and molecular biology The transcription factor Stat3, implicated in cell transformation induced by many oncogenes, is also a downstream signaling molecule activated by HGF/SF-Met signaling. In collaboration with Richard Jove, we have utilized Stat3β, a dominant negative form of Stat3, to show that Stat3 activity is critical both for HGF/SF- Met–mediated cell growth in soft agar and for tumor growth in athymic nude mice. Our current efforts are designed to use Stat3β to identify the downstream targets of Stat3 that influence anchorage-independent growth. Therapeutics In collaboration with David Wenkert, we have been testing novel derivatives of the antitumor agent geldanamycin for suppression of Met activity in cultured tumor cells. We have observed that the plasmin-inhibitory activity of geldanamycin and its derivatives extend over a concentration range of almost nine logs. The most potent compounds inhibited plasmin activity at IC50’s of 5–30 fM, in stark contrast to the nanomolar concentrations required for the destabilizing effects of geldanamycin on HSP90. Three of the derivatives are more potent than geldanamycin: 17-[di-(2- chloroethyl)amino]-17-demethoxygeldanamycin, 17-amino-17-demethoxygeldanamycin, and the most potent, 7′-bromogeldanoxazinone. In vivo imaging In collaboration with Brian Cao, Beatrice Knudsen, and Milton Gross, we are developing monoclonal antibodies raised against components of the Met-HGF/SF receptor–ligand pair as potential diagnostic and therapeutic agents. We 45

have undertaken a detailed characterization of the single anti-Met monoclonal antibody designated Met3. By immunofluorescence we have shown that Met3 binds with high avidity to cultured tumor cells expressing human Met. We have also shown that Met3 binds to cultured normal prostate epithelial cells and to prostate epithelium in human tissue sections. We have confirmed by FACS analysis that Met3 binds to cells of the human prostate carcinoma lines PC- 3 and DU145, which are known to express Met. We have examined the ability of Met3 to image human tumors of different tissue origins. Tumor xenografts were raised subcutaneously in hind limbs of athymic nude mice, and animals were injected intravenously with [ 125 I]Met3. Total-body gamma camera images were acquired and analyzed. The autocrine Met-expressing tumors S-114 (3T3 murine cells transformed with human Met and HGF/SF) and SK-LMS-1 (human leiomyosarcoma) and the paracrine Met-expressing human prostate carcinoma PC-3 were all readily imaged with [ 125 I]Met3; peak tumor-to-control hind limb asymmetry was observed at about 3 days postinjection (Figure 1). Figure 1. Leiomyosarcoma in mouse thigh imaged with radioactive anti-Met monoclonal antibody From these observations we conclude that anti- Met monoclonal antibodies—Met3 in particular— are robust reagents for detecting human Metexpressing cells. They are worthy of further evaluation as potential diagnostic and therapeutic agents for human cancers, including prostate cancer. In collaboration with Brian Ross, our visiting scientists Ilan and Galia Tsarfaty are leading an effort to develop a noninvasive tumor molecular imaging program. In order to study the metabolic effects of Met-HGF/SF signaling in vivo, we recently demonstrated functional molecular imaging of Met receptor activity. DA3 mammary adenocarcinoma cells were injected into the mammary glands of mice, forming tumors expressing high levels of Met. We showed that Met activation in vivo by HGF/SF alters the hemodynamics of normal and malignant Met-expressing tissues. Organs and tumors expressing high levels of Met showed the greatest alteration in blood oxygenation levels as measured by BOLD-MRI (blood oxygenation level–dependent MRI). Met-expressing tumors showed a 30% change in signal by BOLD-MRI, while no significant alteration was observed in tumors or organs that do not express Met. The extent of MRI signal alteration correlated with the dose of HGF/SF administered. In autocrine tumors, the hemodynamic changes in the tumors are greatly enhanced compared with those in tumors that do not express HGF/SF. Moreover, the kidneys and livers of mice bearing autocrine tumors demonstrate increased hemodynamic activity that is proportional to the tumor size. These results indicate that functional molecular imaging of Met expression can serve as a powerful tool for understanding the metabolic activities affected by its signal transduction, and this approach could be used to understand the different molecular mechanisms of receptor activation. Moreover, functional molecular imaging of Met expression may be useful for the detection, analysis, and prognosis of a wide spectrum of human solid tumors. Human pathology collaborative studies In collaboration with Beatrice Knudsen, we have shown an overall 52% Met positivity among primary prostate cancers in a cohort of 90 46

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