Generation Of A Novel Transgenic Mouse Model With Regulatable Beta-Catenin Expression To Examine Importance Of Activated Beta-Catenin Signaling To Long-Term Maintenance Of Mammary Tumor Growth.

Stabilized β-catenin expression is a well described initiator of mammary tumorigenesis in the mouse and elevated nuclear expression has been observed in human triple-negative tumor samples. However, the importance of stabilized β-catenin to continued tumor growth after initiation, and in the context of other driver mutations, has yet to be elucidated. To ascertain the importance of stabilized β-catenin after tumor initiation, we generated a novel transgenic mouse model, utilizing the tet-off system, to control expression of stabilized β-catenin. Expression was tissue restricted through insertion of a stop cassette flanked by loxP sites upstream of stabilized β-catenin and firefly luciferase, which were coexpressed to allow repeated bioluminescent imaging of experimental mice. Insertion of the expression vector was targeted to the Rosa26 locus in R1 embryonic stem cells through homologous recombination. Following aggregation and chimera generation, two founder lines were established. Pups from early litters carrying one allele of regulatable stabilized β-catenin at the Rosa26 locus, but not targeted by Cre, were smaller in size compared with wild-type littermates. They also developed perianal skin lesions around 2-3 weeks of age, with severe necrosis evident by 3-4 weeks of age, requiring the animals to be euthanized. Backcrossing to the desired FVB strain did not alter development of skin lesions, which were observed in offspring from both founder lines. While aberrant gene expression was not observed in these animals, placing breeding females on doxycycline chow through pregnancy, up until pup weaning, eliminated the condition, suggesting a role for the tet machinery in the development of the condition. Maintenance of breeding cages on doxycycline chow allowed for healthy transgenic pups to survive past 3 weeks of age. Initially, six animal cohorts were established, along with control animals, using two different mammary Cre strains, WAP-Cre and MMTV-NLST-Cre, crossed with regulatable, stabilized β-catenin (St-bcatFL) animals, as well as animals expressing mutated p53-R270H. While mammary tumors developed in animals expressing Cre-targeted regulatable St-bcatFL, the penetrance and growth kinetics were lower than observed with other mammary mouse models expressing stabilized β-catenin. As significantly fewer animals developed tumors, we were only able to test the effect of turning off stabilized β-catenin expression following tumor initiation in a small number of animals, with tumor stasis but not regression observed in animals expressing Cre-targeted St-bcatFL, but not in animals expressing both St-bcatFL and p53-R270H. The regulatable St-bcatFL mice were also crossed with animals expressing mutant PIK3CA*E545K and MMTV-NLST-Cre, to examine the importance of stabilized β-catenin to the rapid tumor growth we have observed in mice expressing stabilized β-catenin through deletion of exon 3, and PIK3CA*E545K. While all of the PIK3CA*E545K;Exon3 fl/+; Cre+ animals examined developed multiple mammary tumors by 5 months of age, the PIK3CA*E545K;St-bcatFL;Cre+ mice failed to develop tumors within the same time period. These results indicate that mammary tumor development, including penetrance and growth kinetics, differs significantly between our novel regulatable stabilized β-catenin mouse model and other more aggressive models of stabilized β-catenin. These differences will require alternative methods to determine whether stabilized β-catenin expression is required for the long-term maintenance of tumor growth. Funding for this project was provided by the Terry Fox Foundation, as well as the Canadian Breast Cancer Foundation. Citation Format: Jennifer L. Gorman, Jessica R. Adams, Emma M. Jones, Sean E. Egan, James R. Woodgett. Generation of a novel transgenic mouse model with regulatable β-catenin expression to examine importance of activated β-catenin signaling to long-term maintenance of mammary tumor growth [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A29.