Characterization of mice carrying mutations of BRCA1 interacting proteins

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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.