The long-term goal of this work is to gain a greater understanding of multistep tumorigenesis at the molecular genetic level. Defining the cumulative genetic events that lead to cancer is ultimately necessary to fully understand the neoplastic process and may provide opportunities for the prevention, diagnosis, and rational treatment of this disease. Toward the long-term objective, an effective transgenic mouse model system has been developed that allows identification and isolation of genes involved in multistep mammary tumorigenesis. This model system takes advantage of transgenic mice that already harbor one genetic lesion (an activated Wnt-1 proto-oncogene) but require further mutations for transformation to neoplasia. the general strategy is to use a retrovirus (mouse mammary tumor virus, MMTV) as a mutagen to activate cooperating cellular proto-oncogenes during infection of these mice and thus accelerate tumor development. Because MMTV activates proto-oncogenes by integrating its own genome nearby, the activated genes may be identified and clones based on their typically close proximity to the newly integrated viral DNA. Preliminary studies using this system have already lead to the identification of two known proto-oncogenes and one novel putative proto-oncogene that cooperate with Wnt-1 in tumorigenesis. The first specific aim is to identify the novel (and known) proto- oncogenes that collaborate with Wnt-1 in mammary tumorigenesis. The methods to define the isolate novel cooperating proto-oncogenes are to isolate the clonal viral insertion sites from tumors by molecular cloning in lambda phage, establish that the site cloned is an integration site in at least one other tumor by Southern blot analysis, and locate the gene in this locus by Northern blotting using probes from the cloned locus. New retroviral mutagens that will sensitize the detection of novel proto-oncogenes will be constructed and used. Pairs of proto- oncogenes that are never activated in the same tumors may indicate that they lie in the same biochemical pathway (i.e., are in the same complementation group). The second specific aim is to begin the physical and biological characterization of the novel proto-oncogenes. Characterization of novel genes, including the one already isolated, will consist of isolating and sequencing cDNA and genomic clones, establishing expression patterns by Northern blot analysis, confirming transforming activities in cell culture and in transgenic mice, and analyzing protein using antipeptide antibodies.
