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2008 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests Many malignant sarcomas such as fibrosarcomas are refractory to available treatments. However, sarcomas possess unique vascular properties which indicate they may be more responsive to therapeutic agents that target endothelial function. Mitogen-activated protein kinase kinases (MKKs) have been shown to play an essential role in the growth of carcinomas, and we hypothesize that signaling through multiple MKK pathways is also essential for sarcomas. One objective of our research is to define the role of MKK signaling in the growth and vascularization of human sarcomas and to determine whether agents such as anthrax lethal toxin (LeTx), a proteolytic inhibitor of MKKs, can form the basis of a novel and innovative approach to the treatment of human sarcoma. In the past year, we have made fascinating discoveries that bring us closer to achieving that objective. Yan Ding and Philippe Depeille, postdoctoral fellows in the lab, with the assistance of Elissa Boguslawski, our xenograft technician, had earlier shown that MKKs are active in soft-tissue sarcomas (including Kaposi sarcoma, fibrosarcoma, malignant fibrous histiocytoma, and leiomyosarcoma) and that LeTx can inhibit the in vitro tumorigenic potential of these cells. We believed that the anti-tumoral properties of LeTx primarily stemmed from its ability to substantially decrease the release of many growth factors—notably the pro-angiogenic vascular endothelial growth factor (VEGF)—from tumor cells, leading to a reduction in tumor growth and vascularization. However, our work this year has changed the way we envision this. As an alternative approach to test the requirement for MKK signaling in fibrosarcoma vascularization in vivo, we established a collaboration with Rick Hay (Laboratory of Noninvasive Imaging and Radiation Biology) to monitor tumor perfusion in xenografts using ultrasound imaging in conjunction with injecting contrast ultrasound microbubbles. We found that inhibition of MKK signaling by LeTx caused a rapid and dramatic decrease in tumor perfusion (Figure 1). Follow-up histologic analysis in collaboration with James Resau (Laboratory of Analytical, Cellular, and Molecular Microscopy) showed this decrease in tumor perfusion was caused by increased extravasation, i.e., tumor blood vessels became leaky (Figure 2). This was unexpected, since published studies have shown that withdrawal of VEGF leads to a regression of neovascularization over the course of weeks, not hours. Our failure to observe similar changes in normal endothelium indicates that the survival requirements for normal and tumor endothelium are distinct. Taken together, our results indicate that while MKK activity is required for tumor cell proliferation, it also plays an important role in tumor vascular function. Further studies are required to delineate the events leading to loss of vascular function, as well as the relative contributions of tumor, stromal, and endothelial cells in this response. Figure 1 Figure 1. Ultrasound analysis of the effects of acute MKK inhibition on tumor blood flow. HT-1080 fibrosarcoma xenograft tumors (approximately 100 mm 3 in diameter) were treated with 1 standard dose of either LeTx or inactive LeTx by i.v. injection. Tumor perfusion was evaluated by ultrasound imaging enhanced with contrast microbubbles either immediately prior to treatment or 24 h after treatment. The contrast signals, displayed in the images as green spots, are proportional to the number of microbubbles within the region of interest, which in turn reflects the included volume of flowing blood. 16

VARI | 2008 Currently, Jenn Bromberg-White, a postdoctoral fellow, is following up these studies with an investigation into the roles these same pathways play in other neovascular diseases such as acute macular degeneration. Chih-Shia Lee, a graduate student, is performing a detailed study of the individual contributions of MKK pathways to melanoma survival, and Jaclyn Lynem, our laboratory technician, is investigating the molecular basis of LF inactivation of MKK. Finally, in our longstanding collaboration with Arthur Frankel, Director of the Scott & White Cancer Research Institute in Texas, we are moving forward with preclinical testing of the therapeutic potential of LeTx in the treatment of malignant melanoma. Figure 2 Figure 2. The effect of acute MKK inhibition on xenograft morphology. Mice bearing HT-1080 xenograft tumors were injected i.v. with inactive LeTx (A) or LeTx (B, C). Twenty-four hours later, tumor (A, B) and kidney (C) tissues were formalin-fixed, paraffin-embedded, sectioned, and stained using hemotoxylin and eosin. Images were obtained at 20X; bars represent 50 μm. From left: Ding, Duesbery, Holman, Boguslawski, Lynem, Lee, Bromberg-White 17

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