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. 2013 Apr 11;32(15):1943-9.
doi: 10.1038/onc.2012.211. Epub 2012 Jun 4.

Akt1 inhibits homologous recombination in Brca1-deficient cells by blocking the Chk1-Rad51 pathway

Y Jia  1 , W Song, F Zhang, J Yan, Q Yang
Affiliations

Akt1 inhibits homologous recombination in Brca1-deficient cells by blocking the Chk1-Rad51 pathway

Y Jia et al. Oncogene. .

Abstract

Brca1 deficiency leads to the development of breast cancer. We previously found that Brca1 deficiency activates the Akt oncogenic pathway. Reduced expression of Brca1 was highly correlated with increased activated Akt in human breast cancer samples. Furthermore, activation of Akt1 was involved in Brca1-deficiency-mediated tumorigenesis in mice. Defective homologous recombination (HR) is thought to be a major contributor to tumorigenesis in Brca1 deficiency. Here, we show that Akt1 promotes chromosome instability in Brca1-deficent cells. DNA breaks in Brca1-deficent cells are aberrantly joined into complex chromosome rearrangements by a process dependent on Akt1. Depletion of Akt1 increases HR in Brca1-mutant cells, which is rescued by expression of wild-type, but not mutant Akt1 with deletion of Brca1-binding domain. Mechanistically, activated Akt1 in Brca1-deficient cells impairs Chk1 nuclear localization and subsequently disrupts interaction of Chk1 and Rad51 leading to HR defects. Our results indicate that Brca1 deficiency might activate Akt1 contributing to tumorigenesis through regulation of the Chk1-Rad51 signaling.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Depletion of Akt1 suppresses tumor development and radial chromosome formation in Brca1Δ11/Δ11 MEFs. (a) Depletion of Akt1 suppresses tumor development induced by Brca1Δ11/Δ11 MEFs in mice. After 8–9 weeks implanting Brca1Δ11/Δ11 and MCF7 cells with or without knockdown of Akt1, the tumors became rigid and the volume of tumor ((L × W2)/2) is measured. (b) Depletion of Akt1 suppresses radial chromosome formation in Brca1Δ11/Δ11 MEFs. Radial chromosome structure characteristic of metaphases from Brca1Δ11/Δ11, Akt1-shRNAs and WT MEFs (n = 300) is indicated. (c) Quantitative analysis of radial chromosomes with 5 Gy IR-treatment in the indicated cells.
Figure 2
Figure 2
Deletion of Akt1 restores HR in Brca1-deficient cells. (a) Deletion of Akt1 restores Rad51 foci formation in Brca1Δ11/Δ11 MEFs. Immunofluorescence images show Rad51 foci and DAPI counterstain in MEFs of the indicated genotypes with 5 Gy IR-treatment for 2 h. Active form of Akt1-S473D, but not deletion of HM mutant (ΔHM), rescues Rad51 foci in Brca1Δ11/Δ11 MEFs with Akt1 knockdown (Akt1-sh2, target sequence in 3′UTR). Bottom panel shows the percentage of cells with Rad51 foci (n = 200 counted for each genotype). (b) Deletion of Akt1 restores HR frequency in Brca1Δ11/Δ11 MEFs. Top: Structure of the HR reporter substrate DR-GFP is shown. Bottom: Frequency of HR relative to total transfected cells of the indicated genotypes, as measured with the DR-GFP reporter. A full colour version of this figure is available at the Oncogene journal online.
Figure 3
Figure 3
Akt1 regulates localization of Chk1 leading to radial chromosome formation and HR-defective in Brca1-deficient cells. (a) Akt1 regulates localization of Chk1 in Brca1-deficient cells. Immunofluorescence images show pChk1 localization in the indicated MEFs. (b, c) The Akt1-Chk1 pathway is responsible for radial chromosome formation and HR defect in Brca1-mutant cells. Quantitative analysis of radial chromosomes with 5 Gy IR-treatment in indicated cells (n = 300) (b). Frequency of HR relative to total transfected cells of the indicated genotypes, as measured with the DR-GFP reporter (c). A full colour version of this figure is available at the Oncogene journal online.
Figure 4
Figure 4
Rescue of HR in Brca1-deficient cells by Akt1 depletion correlates with activating the Chk1-Rad51 pathway. (a) Depletion of Chk1 abrogates IR-induced Rad51 foci in Brca1/Akt1-deficient cells. The quantitative data show Rad51 foci in MEFs of the indicated genotypes with 5 Gy IR-treatment for 2 h. (b) Akt1 regulates subcellular localization of Chk1 and Rad51. Nuclear and cytoplasmic lysates prepared from the indicated MEFs were subjected to Western blotting analysis with the indicated antibodies. (c) Akt1 regulates Chk1-Rad51 interaction in Brca1-deficient cells. The nuclear extracts from the indicated MEFs were subjected to immunoprecipitation with anti-Rad51 or IgG antibodies. This was followed by Western blotting to detect pChk1 and Rad51.
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