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2007 Scientific Report

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VARI | 2007 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 and vascularization of carcinomas, and we hypothesize that signaling through multiple MKK pathways is also essential for sarcomas. The objective of our research is to define the role of MKK signaling in the growth and vascularization of human sarcomas and to determine whether inhibition of multiple MKKs by 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 substantial progress in achieving this objective. Yan Ding, a postdoctoral fellow in the lab, and Lisa Orcasitas have shown that MKKs are active in fibrosarcoma and that LeTx can inhibit the in vitro tumorigenic potential of cells derived from human fibrosarcoma. The anti-tumoral properties of LeTx probably stem from its ability to substantially decrease the release of many growth factors, notably the pro-angiogenic vascular endothelial growth factor (VEGF). In vivo, LeTx caused a substantial decrease in both tumor volume and mean vascular density of fibrosarcoma xenografts. These changes also correlated with a decreased level of pro-angiogenic factors, including VEGF. Dr. Ding also found that the ability of LeTx to decrease the release of VEGF was not limited to fibrosarcoma, but was observed in cell lines derived from various sarcomas including malignant fibrous histiocytoma and leiomyosarcoma. These results are consistent with the hypothesis that MKK signaling is required for the growth and vascularization of fibrosarcoma both in vitro and in vivo, and this probably is also true of other types of soft-tissue sarcomas. Similarly, using an endothelial model of Kaposi sarcoma, Philippe Depeille, another postdoctoral fellow, and Elissa Boguslawski showed that in vitro, LeTx 1) decreases proliferation, 2) inhibits tumorigenesis, and 3) dramatically reduces the secretion of angioproliferative cytokines such as VEGF. Furthermore, in vivo, systemic treatment with LeTx inhibits tumor growth and vascularization. These findings support the importance of MKK pathways in the release of angioproliferative cytokines that promote tumor growth and vascularization. Our data suggest that inhibition of MKK signaling may be an effective therapeutic strategy for the treatment of Kaposi sarcoma. 19 In collaboration with Bart Williams’ lab, John Young, our senior technician, and Jennifer Bromberg-White, a postdoctoral fellow, investigated the mechanism of anthrax toxin entry into cells. Together they showed that mice or cells lacking LRP6, or a related protein called LRP5, are still susceptible to anthrax toxin. The discovery that anthrax toxin can enter cells without the help of LRP6 presents a significant challenge to the published models of anthrax toxin function. These findings will help focus the efforts of scientists working on new ways to treat anthrax. In collaboration with Arthur Frankel, director of the Scott & White Cancer Research Institute in Texas, we have also tested the therapeutic potential of LeTx in the treatment of malignant melanoma. Progress to date indicates that melanoma is particularly sensitive to MKK inhibition. This is likely due in part to the fact that more than 80% of melanoma tumors harbor somatic mutations that cause constitutive activation of the MKK1 and MKK2 signaling pathways, though indirect evidence suggests that other MKK pathways also play a role in melanoma progression. Chih-Shia Lee is performing a detailed study of the individual contributions of MKK pathways to melanoma survival. Jaclyn Lynem and Naomi Asantewa-Sechereh are investigating the molecular basis of LF inactivation of MKK. We are currently performing preclinical studies to evaluate the potential of LeTx as a therapeutic for malignant melanoma.

Van Andel Research Institute | Scientific Report 20 From left: Asantewa-Sechereh, Orcasitas, Lynem, Boguslawski, Lee, Bromberg-White, Duesbery, Holman, Young, Depeille Recent Publications Young, J.J., J.L. Bromberg-White, C.R. Zylstra, J. Church, E. Boguslawski, J. Resau, B.O. Williams, and N. Duesbery. In press. LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin. PLoS Pathogen. Depeille, P.E., Y. Ding, J.L. Bromberg-White, and N.S. Duesbery. 2007. MKK signaling and vascularization. Oncogene 26(9): 1290–1296. Abi-Habib, Ralph J., Ravibhushan Singh, Stephen H. Leppla, John J. Greene, Yan Ding, Bree Berghuis, Nicholas S. Duesbery, and Arthur E. Frankel. 2006. Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma tumors in athymic nude mice. Clinical Cancer Research 12(24): 7437–7443. Bodart, Jean-François L., and Nicholas S. Duesbery. 2006. Xenopus tropicalis oocytes: more than just a beautiful genome. In Xenopus Protocols: Cell Biology and Signal Transduction, X. Johné Liu, ed. Methods in Molecular Biology series, Vol. 322. Totowa, N.J.: Humana Press, pp. 43–53.

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