TUMORIGENESIS IN MICE LACKING OSTEOPONTIN

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Osteopontin (OPN) is a secreted phosphoprotein which binds both to cells and to mineralized surfaces. There is considerable circumstantial and direct evidence indicating that OPN participates in the process of tumorigenesis. We have developed mice which contain a targeted disruption of the OPN gene, and are unable to express osteopontin protein. These mice are viable and fertile. The goal of the research described in this proposal is to use these mice to define the role at the molecular and physiological level that OPN plays in tumorigenesis. We will derive mouse embryo fibroblast lines from mice which can and cannot express OPN, and transform there cells by transfection with different oncogenes. The ability of the cells of differing ON status to grow in soft agar and form tumors in nude mice will be assessed. Others have shown that transformed cells expressing antisense RNA to OPN are impaired in their ability to grow in soft agar and to form tumors, so we expect to see a similar phenotype in cells from the OPN knockout mice. The defect in these cells will be corrected by transfection with OPN expression constructs. To begin to define the molecular mechanism of OPN's action, the OPN deficient cells will be transfected with constructs expressing mutant forms of OPN, and the effects of these mutants on different aspects of tumorigenesis will be correlated with their activity in several in vitro assays. To define the role of OPN in tumorigenesis at the physiological level, we will breed the OPN deficient mice with transgenic mice in which tumors arise rapidly and with high frequency. The kinetics and frequency of tumor formation in animals which express the transgenes but are unable to express OPN will be assessed, and compared with that in animals that do express OPN. Finally, we will address the question of the localization of OPN in tumors. Most normal tissues which express OPN at similar levels to that found in some tumors secrete the protein into a fluid, such as urine or milk. Thus, it is not clear where the OPN made by tumors is, To answer this question, we will develop new, high affinity antibodies to mouse OPN, and verify their specificity using the OPN -/- mice. We will use these antibodies to develop an ELISA to measure mouse OPN in different fluids, and to perform immunohistochemistry on tumors and mouse tissues.