Human cancers contain multiple mutations and it is uncertain which mutations are necessary and sufficient, which are passive passenger mutations, and whether there are other yet undiscovered mutations crucial for transformation. One prospective approach to better understand the roles of specific mutations is to engineer these mutations in normal cells. We propose to engineer tumors in mice by sporadically and somatically creating """"""""mutant"""""""" stem cells with defined genotypes. Unlike most mice models, our approach will better mimic sporadic tumorigenesis because Cre-mediated inactivation of floxed tumor suppressor genes (TSGs) will stochastically occur in isolated, widely scattered single cells. A nonfunctional out-of-frame (An+l) germline Cre-allele will become activated (An+l to An) in a DNA mismatch repair deficient background and subsequently recombine floxed genes to create well-defined mutant genotypes. A current An+l transgenic Cre allele will be targeted to the ubiquitously expressed ROSA26 locus to better control its activation. Similar to sporadic tumorigenesis, stochastic and somatic mutations will occur in single cells surrounded by normal cells. Our studies will examine sporadic Apc, Pten, or Trp53 inactivation in the intestines. Because it is uncertain how many mutations are required for transformation, a floxed B-galactosidase allele will be included so that """"""""mutant"""""""" cells may be identified regardless of resultant phenotype. Certain TSGs may modulate stem cell survival, and even though loss of a single TSG may not confer a neoplastic phenotype, stem cell survival may be increased, resulting in greater numbers of blue staining """"""""mutant"""""""" cells in otherwise normal appearing intestines. Multistep tumor progression implies that cancers arise after a series of mutations. These studies will prospectively recreate this process and define the fates of single stem cells in normal mammalian tissues that acquire one or more concomitant gene alterations. The success or failure of certain mutations to confer neoplastic phenotypes will challenge or confirm canonical tumor progression models.
| Miller, Ashleigh J; Dudley, Sandra D; Tsao, Jen-Lan et al. (2008) Tractable Cre-lox system for stochastic alteration of genes in mice. Nat Methods 5:227-9 |
| Tsao, Jen-Lan; Dudley, Sandra; Kwok, Brian et al. (2002) Diet, cancer and aging in DNA mismatch repair deficient mice. Carcinogenesis 23:1807-10 |
