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2005 Scientific Report

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  • Report
  • Institute
  • Mice
  • Proteins
  • Signaling
  • Protein
  • Michigan
  • Tumor
  • Molecular
  • Laboratory

Melanoma Melanoma is the

Melanoma Melanoma is the most rapidly increasing malignancy among young people in the United States. If detected early, the disease is easily treated, but once the disease has metastasized it has a high mortality rate. Current systemic therapy for advanced, metastatic melanoma includes dacarbazine (DTIC) chemotherapy, either alone or in combination with other agents, and biological therapy using recombinant interferon-α (IFN-α) and/or interleukin-2 (IL-2). However, except on rare occasions, none of these treatments has produced long-term control of the disease, and cytokine therapy is associated with significant toxicities. We have developed a mouse model of melanoma based on the avian RCAS/TVA system. In this model, the retroviral receptor TVA is expressed under the control of the tyrosinaserelated protein 2 (TRP2) promoter, which is expressed in melanocytes. In replicating mammalian cells that express TVA, the viral vector is capable of stably integrating into the DNA and expressing the experimental gene at high levels, but the virus is replication-defective because viral RNA and proteins are inefficiently produced. Therefore, the viral vectors cannot spread in the target animals; in addition, since little viral envelope protein is produced, there is no interference to superinfection. Theoretically, there is no limit to the number of experimental genes that can be introduced into a TVA-expressing mammalian cell. The ability of these cells to be infected by multiple viruses allows efficient modeling of melanoma, because multiple oncogenic alterations can be introduced into the same cell or animal without the costs associated with mating multiple strains of transgenic mice. Activated ras oncogenes, which turn on mitogen-activated protein kinase (MAPK) signaling, are detected in approximately 20% of human melanomas. Recently, activating mutations in the B-raf gene, which also activate MAPK signaling, have been found in more than 65% of malignant melanomas. With mutually exclusive mutations in ras and B-raf, the MAPK signaling pathway is constitutively activated in over 85% of cases of malignant melanoma, indicating its importance. Overexpression of activated H-ras (G12V) specifically in melanocytes of Ink4adeficient mice results in the development of multiple spontaneous cutaneous melanomas. However, unlike in the human disease, these tumors fail to metastasize. A conditional Ink4a/Arf knockout allele, Ink4a-lox, has been introduced into the germline of FVB/N mice. The lox sites flank exons 2 and 3 of this locus such that Cremediated excision eliminates both p16 INK4A and p19 ARF . We have recently obtained two homozygous Ink4a-lox/lox breeding pairs, which will be crossed to our TRP2-TVA transgenic mice. We plan to use the mice to study the role of different genes in melanoma initiation, maintenance, and progression. Glioblastoma Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. It is also the most fatal: mean survival is less than one year from the time of diagnosis, with less than 10% survival after two years. Despite major improvements in imaging, radiation, and surgery, the prognosis for patients with this disease has not changed in the last 20 years. Recently, genes that are differentially expressed in tumor tissue relative to normal brain tissue have been found. However, those that can be productively targeted for therapeutic intervention in human patients remain to be identified. A mouse model of human GBM based on the avian RCAS/TVA system has been developed by Eric Holland. In this model, the retroviral receptor TVA is expressed under the control of the Nestin promoter, which is active in neural and glial progenitors. Intracranial infection of Nestin-TVA mice with RCASBP(A)-Akt and RCASBP(A)- KRas induces glioblastomas that are histologically similar to human GBM. We have generated an RCANBP(A)TRE-KRas virus in which expression of K-Ras can be controlled post-delivery. We are using the Nestin-TVA model to test the function of this virus in vivo. We plan to use these vectors to determine if K-Ras expression is required for tumor maintenance in this system and to identify which genes are appropriate targets for therapy. Antiviral strategies A second, related focus of our group is the identification of effective antiviral strategies using the RCASBP/RCANBP family of retroviral vectors as a model system. Viral diseases pose a 32

major risk to the food supply and to animal welfare, especially in today’s high-intensity animal agriculture. Many viruses are highly communicable and are capable of rapid mutation to escape immune surveillance; few effective antiviral drugs are available. Over the past several years, we have developed strategies aimed at conferring dominant resistance to viral pathogens in chickens. These strategies have focused on manipulating viral (“pathogen-derived resistance”) and/or cellular genes (“host-derived resistance”) to express new proteins capable of disrupting the viral life cycle. The results have provided valuable information on the biology of avian viruses, as well as having the potential for practical application. We are now working to adapt the new RNA interference (RNAi) technology to the development of new anti-viral strategies for two important chicken pathogens, ALV and Marek’s Disease virus (MDV). These two targets have been chosen because both are economically important and because they have distinctly different infectious cycles that provide different challenges for RNAi. External Collaborators Jerry Dodgson, Michigan State University, East Lansing Henry Hunt and Huanmin Zhang, Avian Disease and Oncology Laboratory, East Lansing, Michigan Recent Publications Ai, Minrong, Sheri L. Holmen, Wim van Hul, Bart O. Williams, and Matthew W. Warman. 2005. Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass–associated missense mutations in LRP5 affect canonical Wnt signaling. Molecular and Cellular Biology 25(12): 4946–4955. Holmen, Sheri L., Scott A. Robertson, Cassandra R. Zylstra, and Bart O. Williams. 2005. Wnt-independent activation of ß-catenin mediated by a Dkk-1-Frizzled 5 fusion protein. Biochemical and Biophysical Research Communications 328(2): 533–539. Holmen, Sheri L., Cassandra R. Zylstra, Aditi Mukherjee, Robert Sigler, Marie-Claude Faugere, Mary Bouxsein, Lianfu Deng, Thomas Clemens, and Bart O. Williams. 2005. Essential role of ß-catenin in postnatal bone acquisition. Journal of Biological Chemistry 280(22): 21162–21168. Sanchez-Perez, Luis, Timothy Kottke, Rosa Maria Diaz, Atique Ahmed, Jill Thompson, Heung Chong, Alan Melcher, Sheri Holmen, Gregory Daniels, and Richard G. Vile. 2005. Potent selection of antigen loss variants of B16 melanoma following inflammatory killing of melanocytes in vivo. Cancer Research 65(5): 2009–2017. Bromberg-White, Jennifer L., Craig P. Webb, Veronique S. Patacsil, Cindy K. Miranti, Bart O. Williams, and Sheri L. Holmen. 2004. Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. Journal of Virology 78(9): 4914–4916. Holmen, Sheri L., Troy A. Giambernardi, Cassandra R. Zylstra, Bree D. Buckner-Berghuis, James H. Resau, J. Fred Hess, Vaida Glatt, Mary L. Bouxsein, Minrong Ai, Matthew L. Warman, and Bart O. Williams. 2004. Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. Journal of Bone and Mineral Research 19(12): 2033–2040. From left to right: Russo, Holmen 33

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