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2002 Scientific Report

  • Text
  • Institute
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  • Tumors
  • Protein
  • Signaling
  • Michigan
  • Molecular
  • Proteins
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ackgrounds to assess

ackgrounds to assess whether alleles that may modify bone density and lens vascularization (and perhaps Wnt signaling) can be identified in the mouse genome. In addition, we are assessing whether Lrp5 is required for mammary tumorigenesis in MMTV-Wnt1 transgenic mice. Analysis of mice deficient for both Lrp5 and Lrp6 Mice carrying a mutated allele of Lrp6 (generously provided by Bill Skarnes) are being crossed to mice deficient for Lrp5. We are interested in potential phenotypes of Lrp6+/–;Lrp5–/– mice. One possibility is that these mice will have more severe defects in bone density and eye vascularization. In addition, we are determining the phenotype of mice homozygously deficient for both genes. We expect these mice will die very early in gestation. To further characterize the functions of these genes, we are creating mouse embryonic stem cell lines deficient for both genes and using them to generate chimeric mice for analysis (in collaboration with the VARI laboratory of Pamela Swiatek). Wnt-Fz fusion constructs and specificity in the Wnt signaling pathway We have recently published an analysis of fusion proteins between selected Wnt and Frizzled molecules in collaboration with Adrian Salic and Marc Kirschner of Harvard Medical School. We found that expression of several such fusion proteins with Lrp6 could significantly activate a Wnt/β-catenin–responsive reporter gene and stabilize cytoplasmic levels of β-catenin. We are continuing to utilize these constructs to address questions about Wnt signaling specificity. Expression of Wnt receptor components in tumorigenesis and development We have developed probes for RT-PCR analysis of Wnt, Frizzled, Lrps, Dkks, Kremens, and FRPs. We are systematically examining the expression of these components in various tumor cell lines and in tissue samples. Mouse models for melanoma Our laboratory is also interested in the broad goal of improving mouse models of carcinogenesis. Given our interest in Wnt signaling, we are particularly focused on developing models in which alterations in this signaling pathway have been introduced into the mouse genome. An in vivo model is being developed for studying melanoma using a retroviral-based gene targeting system. We have generated a mouse strain expressing the avian leukosis virus receptor, TV-A, under the control of a promoter that is only active in melanocyte precursor cells. These cells can then be infected by avian leukosis virus A (AVL-A) which is harboring genes of interest that have been introduced into AVL-A vectors. This system allows multiple genes to be delivered to a single TV-A+ cell. We can then study the effects of multiple oncogenes and tumor suppressor genes on melanoma development and progression. In addition, this model will allow comparison of melanomas induced by different genetic changes. These can then be used to evaluate the efficacy of different therapeutic strategies. We are also developing a system to regulate the expression of an activated version of β-catenin in melanocytes. Given that mutations in the β- catenin gene have been identified in melanomas, we feel that this system will allow insight into the role of Wnt signaling in melanocyte differentiation and melanoma-genesis. The role of Wnt signaling in prostate cancer We are developing a tetracycline-regulated system to control the expression of an activated version of β-catenin specifically in the prostate. The rationale for doing this is that up-regulation of β-catenin activity is observed in a significant percentage of prostate tumors. In addition, the β- catenin protein physically interacts with the androgen receptor and alters its activity. This work is done with in collaboration with Wade Bushman of the University of Wisconsin. The role of MMP8 in melanoma Expression of matrix metalloproteinase 8 (MMP8), previously thought to be restricted in expression to neutrophils, was recently detected in melanomas but not normal melanocytes. In addition, expression was also detected in neural crest cells during embryonic development. We are currently creating mice deficient in MMP8 to further define its role in development. We are also examining various stages of melanoma to identify the point in melanoma progression at which MMP8 is turned on. 57

External Collaborators Wade Bushman, University of Wisconsin – Madison J. Fred Hess, Merck Pharmaceuticals Marc Kirschner and Adrian Salic, Harvard Medical School, Boston, Massachusetts Publications Holmen, Sheri L., Adrian Salic, Cassandra R. Zylstra, Marc W. Kirschner, and Bart O. Williams. In press. A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate β- catenin-dependent signaling. Journal of Biological Chemistry Yang, Hong, Bart O. Williams, Phillip W. Hinds, T. Shane Shih, Tyler Jacks, Roderick T. Bronson, and David M. Livingston. 2002. Tumor suppression by a severely truncated species of the retinoblastoma protein. Molecular and Cellular Biology 22(9): 3103–3110. From left to right, back row: Williams, Daugherty, Robertson, Zylstra middle row: Giambernardi, Holmen front row: Charbonneau, Mieras 58

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