Characterization of mice carrying mutations of BRCA1 interacting proteins

在过去的一年里，我们发表了我们对Aurora-A和Wee 1的研究。 I. Aurora-A在遗传稳定性、中心体复制和乳腺肿瘤发生中的作用 Aurora-A/STK 15/BTAK编码中心体相关激酶，在90%的乳腺癌中扩增和过表达。先前的一项研究表明，Aurora-A与BRCA 1相互作用并磷酸化。然而，由于从不同的实验系统获得的数据相互矛盾，Aurora-A的过度表达和肿瘤发生之间的因果关系尚未完全建立。为了研究这一点，我们产生了携带MMTV-Aurora-A转基因的小鼠品系。我们发现，所有的MMTV-Aurora-A小鼠显示增强的分支形态发生在乳腺和约40%的发展乳腺肿瘤在20个月大。肿瘤发生率在p53（+/-）突变背景下显著增加，约70% MMTV-Aurora-A;p53（+/-）动物在18月龄时发生肿瘤。值得注意的是，Aurora-A的过表达导致遗传不稳定性，其特征在于在肿瘤形成之前的阶段的中心体扩增、染色体四倍化和过早的姐妹染色单体分离。最值得注意的是，由于AKT通路的激活，包括磷酸化AKT和雷帕霉素的哺乳动物靶蛋白水平升高，以及细胞周期蛋白D1的核积累，这使得四倍体细胞能够持续增殖，严重的染色体异常不会导致细胞死亡。这些数据证实Aurora-A是一种致癌基因，通过诱导遗传不稳定性和激活致癌途径（如AKT及其下游信号传导）导致恶性转化。 二.细胞周期关卡中的Wee 1与遗传稳定性 Wee 1是BRCA 1的下游转录靶点。Wee 1激酶通过磷酸化和失活有丝分裂细胞周期蛋白依赖性激酶1（Cdk 1）来调节G2/M细胞周期检查点。在许多系统中，包括酵母和果蝇，Wee 1的丢失导致过早的有丝分裂进入。然而，Wee 1在哺乳动物中的发育作用仍不清楚。本研究通过基因打靶的方法建立了Wee 1基因敲除小鼠。我们发现Wee-/-胚胎在γ射线诱导的G2/M细胞周期检查点有缺陷，并在胚胎（E）3.5天前死于凋亡。为了进一步研究Wee 1的功能，我们开发了MEF细胞，其中Wee 1被他莫昔芬诱导的Cre-LoxP方法破坏。我们发现Wee 1的急性缺失导致严重的生长缺陷和细胞死亡。Wee 1缺陷细胞显示染色体非整倍性和DNA损伤，如γ-H2 AX灶形成和Chk 2激活所揭示的。进一步的研究揭示了Wee 1在有丝分裂进入和G2/M检查点调控中的保守机制。这些数据提供了体内证据表明，哺乳动物Wee 1在维持基因组完整性方面起着关键作用，并且对于小鼠发育的植入前阶段的胚胎存活至关重要。我们目前正在使用Cre-LoxP方法研究Wee 1在乳腺肿瘤发生中的作用。

In the past year, we have published our studies on Aurora-A and Wee1. I. Aurora-A in genetic stability, centrosome duplication and mammary tumorigenesis Aurora-A/STK15/BTAK, which encodes a centrosome-associated kinase, is amplified and overexpressed in 90% of breast cancer. A previous study indicated that Aurora-A interacts and phosphorylates BRCA1. However, the causal relationship between overexpression of Aurora-A and tumorigenesis has not been fully established due to contradictory data obtained from different experimental systems. To investigate this, we generated a mouse strain that carries an MMTV-Aurora-A transgene. We showed that all the MMTV-Aurora-A mice displayed enhanced branch morphogenesis in the mammary gland and about 40% developed mammary tumors at 20 months of age. The tumor incidence was significantly increased in a p53(+/-) mutation background with about 70% MMTV-Aurora-A;p53(+/-) animals developed tumors at 18 months of age. Of note, overexpression of Aurora-A led to genetic instability, characterized by centrosome amplification, chromosome tetraploidization and premature sister chromatid segregation, at stages prior to tumor formation. Most notably, the severe chromosomal abnormality did not cause cell death owing to the activation of AKT pathway, including elevated levels of phosphorylated AKT and mammalian target of rapamycin, and nuclear accumulation of cyclin D1, which enabled continuous proliferation of the tetraploid cells. These data establish Aurora-A as an oncogene that causes malignant transformation through inducing genetic instability and activating oncogenic pathways such as AKT and its downstream signaling. II. Wee1 in cell cylce checkpoint and genetic stability Wee1 is a downstream transcriptional target of BRCA1. Wee1 kinase regulates the G2/M cell cycle checkpoint by phosphorylating and inactivating the mitotic cyclin-dependent kinase 1 (Cdk1). Loss of Wee1 in many systems, including yeast and drosophila, leads to premature mitotic entry. However, the developmental role of Wee1 in mammals remains unclear. In this study, we established Wee1 knockout mice by gene targeting. We found that Wee-/- embryos were defective in the G2/M cell cycle checkpoint induced by gamma-irradiation and died of apoptosis before embryonic (E) day 3.5. To study the function of Wee1 further, we have developed MEF cells in which Wee1 is disrupted by a tamoxifen inducible Cre-LoxP approach. We found that acute deletion of Wee1 resulted in profound growth defects and cell death. Wee1 deficient cells displayed chromosome aneuploidy and DNA damage as revealed by gamma-H2AX foci formation and Chk2 activation. Further studies revealed a conserved mechanism of Wee1 in regulating mitotic entry and the G2/M checkpoint compared with other lower organisms. These data provide in vivo evidence that mammalian Wee1 plays a critical role in maintaining genome integrity and is essential for embryonic survival at the pre-implantation stage of mouse development. We are currently studying the role of Wee1 in mammary tumorigenesis using the Cre-LoxP approach.