siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors.
siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors.
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Trp 53杂合子BALB/c小鼠发生高比例的自发性乳腺肿瘤，这是一种不同于其他小鼠品系的表型。因此，BALB/c-Trp 53 +/−雌性小鼠类似于以早发乳腺癌为特征的遗传性Li-Fraumeni综合征（LFS），尽管LFS涉及TP 53突变，这可能不仅涉及功能丧失，还涉及功能获得。先前对BALB/c-Trp 53 +/−雌性动物肿瘤的分析显示，Trp 53野生型等位基因的杂合性丢失频繁，并显示出有丝分裂重组的特征。DNA双链断裂（DSB）修复功能障碍，特别是同源重组（HR）的关键参与，也被注意到在人类乳腺癌的病因学。为了更好地定义BALB/c-Trp 53 +/−小鼠中的功能改变，我们对来自BALB/c-Trp 53 +/−与C57 BL/6 J-Trp 53 +/−小鼠的小鼠胚胎成纤维细胞（MEF）应用了基于荧光的DSB修复试验。这种方法揭示了在BALB/c-Trp 53 +/−中HR的失调，但不是非同源末端连接（NHEJ），这在乳腺上皮细胞中得到了进一步证实。筛选靶向DSB修复、重组、复制和信号传导基因的小干扰RNA文库，鉴定了25个基因，其在沉默后引起两种菌株中同源DSB修复之间的差异。相互作用组分析的命中揭示集群的复制相关和范可尼贫血（FA）/乳腺癌易感性（BRCA）基因。通过核53 BP 1、复制蛋白A（RPA）和Rad 51灶的免疫荧光显微镜进一步解剖BALB/c-Trp 53 +/−中的功能变化，发现交联和复制相关修复的差异。染色体断裂、G2期阻滞和生化分析表明，FancD 2下游的FA途径缺陷与BRCA 2水平降低相关。与BRCA的多基因模型一致，因此，在p53部分功能丧失的背景下，FA通路中的BRCA修饰等位基因可能促进BALB/c-Trp 53 +/−小鼠的乳腺癌发生。
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.
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