siRNA screening identifies differences in the Fanconi anemia pathway in BALB|[sol]|c-Trp53|[plus]||[sol]||[minus]| with susceptibility versus C57BL|[sol]|6-Trp53|[plus]||[sol]||[minus]| mice with resistance to mammary tumors

摘要

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/? female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/? females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/? mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/? versus C57BL/6J-Trp53+/? mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/?, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/? by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/? mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.