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2006 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Melanoma Activated NRAS oncogenes, which turn on mitogen-activated protein kinase (MAPK) signaling, are detected in approximately 20% of human melanomas. Recently, activating mutations in the BRAF gene, which also activate MAPK signaling, have been found in more than 65% of malignant melanomas. With mutually exclusive mutations in RAS and BRAF, the MAPK signaling pathway is constitutively activated in over 85% of cases of malignant melanoma, indicating its importance. Interestingly, it was recently observed that tumors harboring BRAF mutations are much more sensitive to pharmacological inhibition of downstream MAPK signaling than those with NRAS mutations. This suggests that single-agent therapeutic strategies may be ineffective in tumors containing RAS mutations and that rational combination therapeutic strategies will be required. The RAS subfamily consists of H (Harvey) RAS, N (neuroblastoma) RAS, and two splice variants of K (Kirsten) RAS (KRAS4A and KRAS4B). In many tumors, oncogenic mutations have been identified at positions 12, 13, or 61, which cause RAS to remain constitutively active. With the exception of thyroid cancers, most tumors are associated with mutation of only one isoform of RAS and this association cannot be explained solely by differential regulation of RAS gene expression in different tissues. HRAS mutations are more commonly associated with bladder and kidney cancers; KRAS mutations are found in lung, colorectal, ovarian, and pancreatic cancers; and NRAS mutations are most commonly associated with melanoma and hematologic malignancies. 40 We have been characterizing the transforming capabilities of the different Ras isoforms in a nontransformed, immortal Ink4a/Arfdeficient mouse melanocyte cell line. We have observed that activated NRas is able to transform these cells much more efficiently than either activated HRas or KRas; and whereas expression of NRas increases the proliferation of the melanocytes, expression of KRas does not. Furthermore, coexpression of c-myc with KRas in these cells mimics the proliferation and transformation capabilities of NRas alone, whereas coexpression of Akt with KRas in these cells mimics the proliferation and transformation capabilities of HRas alone. These data suggest that the different Ras isoforms have distinct, nonredundant functions in melanocytes and may explain why most melanomas are associated with mutation of only one isoform of Ras. 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. Malignant gliomas can be induced in these mice through the combined expression of activated forms of both KRas and Akt in glial progenitor cells. To determine the reliance of these tumors on continued KRas signaling in vivo, we generated a viral vector that allows the expression of KRas to be controlled post-delivery. Tumor-free survival rates were compared between those animals with continued KRas expression and animals in which KRas expression was suppressed. KRas signaling was found to be required for the maintenance of these tumors in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and increased survival. Subsequent reexpression of KRas reinitiated tumor growth, indicating that a percentage of the progenitor cells survived and retained tumorigenic properties. This model shows the crucial importance of the Ras pathway in glioblastoma maintenance and indicates that continuous suppression of Ras signaling is necessary and sufficient to suppress the tumorigenic potential of the glial progenitor cells. In addition, this regulated expression system will allow the evaluation of the role of other genes and pathways in this context. This has important clinical implications for pharmacologic agents targeting these pathways in GBM patients.

VARI | 2006 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 major risk to the food supply and to animal welfare, especially in today’s highintensity 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 antiviral strategies for two important chicken pathogens, avian leukosis virus and avian influenza. 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 John Brigande, Oregon Health & Science University, Portland Jerry Dodgson, Michigan State University, East Lansing Henry Hunt and Huanmin Zhang, Avian Disease and Oncology Laboratory, East Lansing, Michigan Nita Maihle, Yale University School of Medicine, New Haven, Connecticut Phillipe Monnier, University of Toronto, Canada Bill Pavan, National Institutes of Health, Bethesda, Maryland Maria Soengas, University of Michigan, Ann Arbor Richard Vile, Mayo Clinic, Rochester, Minnesota 41 Recent Publications From left to right, rear: Whitwam, Holmen; front: Russo, Warlick Park, Ki-Sook, Soung Hoo Jeon, Sung-Eun Kim, Young-Yil Bahk, Sheri L. Holmen, Bart O. Williams, Kwang-Chul Chung, Young-Joon Surh, and Kang-Yell Choi. 2006. APC inhibits ERK pathway activation and cellular proliferation induced by Ras. Journal of Cell Science 119(5): 819–827. Wang, PengFei, Dong Kong, Matthew W. VanBrocklin, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams, and Sheri L. Holmen. 2006. Simplified method for the construction of gene targeting vectors for conditional gene inactivation in mice. Transgenics 4: 215–228. Holmen, Sheri L., and Bart O. Williams. 2005. Essential role for Ras signaling in glioblastoma maintenance. Cancer Research 65(18): 8250–8255.

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