Abstract 3020: Homology-directed repair in mouse mammary tissue

Abstract

Abstract Numerous factors are linked to breast cancer risk including reproductive history, early exposure to radiation, age and genetic background. Defects in homology-directed repair (HDR)_considered the most accurate of the three major pathways for repairing double-strand breaks (DSBs) in DNA in mammalian cells_can lead to genomic instability and are associated with tumor predisposition. Mutations in multiple genes involved in HDR are linked to breast cancer susceptibility including BRCA1 and BRCA2. The mammary gland is highly influenced by its hormonal environment and undergoes significant changes during pregnancy, lactation and involution, but many questions remain about how the DNA damage response in mammary tissue, and specifically DNA DSB repair, is impacted by these developmental changes. A widely used reporter for studying DSB repair by HDR is DR-GFP in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats, resulting in a functional GFP gene. We have recently generated DR-GFP containing mice that express I-SceI under the control of a tetracycline-inducible promoter. Induction of I-SceI by doxycycline administration ex vivo results in substantially increased HDR in primary cultures of mammary epithelial cells compared to transient transfection experiments. To gain insight into the effects of breast cancer predisposing mutations on HDR in mammary epithelial cells, the I-SceI/DR-GFP mice were crossed to mice carrying hypomorphic mutations in Brca1 (Brca1tr/tr) or Brca2 (Brca2ex27/ex27). An approximately 3-fold reduction in HDR was observed in primary mammary epithelial cells from virgin Brca1tr/tr I-SceI DR-GFP mice compared to littermate controls, while an 8-fold reduction was observed in primary mammary epithelial cells from virgin Brca2ex27/ex27 mice. Primary ear fibroblasts from these animals showed similarly reduced HDR levels. In order to understand how DSB repair processes in the mammary gland are affected by developmental changes we are assessing HDR at different stages of mammary gland development in vivo. We observe a significant increase in HDR relative to overall repair in mammary tissue from mice at mid-pregnancy compared to virgin littermates. We are now attempting to determine if there is a relationship between repair proficiency, lineage specificity of mammary epithelial cells and fate after involution in mammary tissue from normal and BRCA mutant mice. Citation Format: Elizabeth Kass, Mary Ellen Moynahan, Maria Jasin. Homology-directed repair in mouse mammary tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2015-3020