The outcome of this SBIR application will be a rat model of colorectal cancer (CRC), which can be used to discover new targets for colon cancer therapy. CRC rats will be produced using Transposagen's novel technology for introducing somatic mutations in rats. A number of germline and somatic mutations are known to contribute to the initiation and progression of both heritable and sporadic CRC, but identifying the causative molecular and genetic defects involved in human CRC has proven difficult. Existing mouse models for CRC do not accurately model human disease. Rats more closely recapitulate the molecular, cellular, and pathologic characteristics of human CRC. Polyps in the rat, unlike in the mouse, progress to adenocarcinomas that are equivalent to stage T1 CRC in humans. Colon cancer induced in normal rats using known human carcinogens will spread to the liver, indicating that the rat, unlike the mouse, may serve as a valuable model for CRC metastasis. The physical size of the rat also makes it much more amenable to endoscopy, thus enabling polyp monitoring and real-time assessment of somatic mutations taking place in a single adenoma, so that gate-keeper mutations can be more accurately discriminated from mutations involved in later stages of carcinogenesis. In Phase I of this application, we will create and characterize three lines of transgenic rats: 1) rats exhibiting intestine-specific expression of a Cre recombinase using a VilCre transgene;2) rats expressing a transposase (the enzyme responsible for transposon mobilization) gene under the direction of the ubiquitous ROSA26 promoter. A lox-stop-lox cassette will be used to restrict the expression of the transposase;3) transgenic rats containing a gene trap donor transposon. Cre expression in tri-transgenic rats, created by cross-breeding, will trigger expression of the transposase, which will then mobilize the gene trap transposon and insert it randomly throughout the genome. Importantly, these mutations will occur in the intestine only, because of the tissue-specific expression of the Cre recombinase. Our gene-trap will be capable of creating both null alleles (loss-of-function) and over-expressed transcripts (gain-of-function). A sequence-specific tag is used for mapping mutations, an especially salient advantage compared to other random mutagenesis methods (such as chemical or radiation strategies) for rapid identification of tumor suppressors and oncogenes. In later Phase II work, we will perform a full phenotypic analysis of the CRC phenotype of tri-transgenic rats and clone insertion sites to verify the efficacy of the model. The CRC rat model developed by our method of unbiased transposon mutagenesis in the intestine will likely identify novel cancer genes in a model of colon cancer that is more relevant to human CRC.
Rats recapitulate the dietary, genetic, and pathological characteristics of human colon cancer more accurately than mice. In addition, rats are more amenable to colonoscopy and biopsies because of their size, and therefore, are more convenient than mice for finding and studying the polyps and pre-cancerous lesions that lead to colon cancer. Here we propose a strategy using mobile DNA (or jumping gene) technology in this more relevant animal model to rapidly discover new cancer genes, which may ultimately lead to better assessment of genetic risk, earlier diagnosis, and new cancer drugs for humans.
