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. 2015 Jun 4;34(23):3023-35.
doi: 10.1038/onc.2014.239. Epub 2014 Aug 4.

WEE1 murine deficiency induces hyper-activation of APC/C and results in genomic instability and carcinogenesis

A Vassilopoulos  1 , Y Tominaga  2 , H-Seok Kim  3 , T Lahusen  2 , B Li  4 , H Yu  4 , D Gius  5 , C-X Deng  2
Affiliations

WEE1 murine deficiency induces hyper-activation of APC/C and results in genomic instability and carcinogenesis

A Vassilopoulos et al. Oncogene. .

Abstract

The tyrosine kinase WEE1 controls the timing of entry into mitosis in eukaryotes and its genetic deletion leads to pre-implantation lethality in mice. Here, we show that besides the premature mitotic entry phenotype, Wee1 mutant murine cells fail to complete mitosis properly and exhibit several additional defects that contribute to the deregulation of mitosis, allowing mutant cells to progress through mitosis at the expense of genomic integrity. WEE1 interacts with the anaphase promoting complex, functioning as a negative regulator, and the deletion of Wee1 results in hyper-activation of this complex. Mammary specific knockout mice overcome the DNA damage response pathway triggered by the mis-coordination of the cell cycle in mammary epithelial cells and heterozygote mice spontaneously develop mammary tumors. Thus, WEE1 functions as a haploinsufficient tumor suppressor that coordinates distinct cell division events to allow correct segregation of genetic information into daughter cells and maintain genome integrity.

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Conflict of interest statement

Conflict of Interest: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Wee1 mutant cells display mitotic aberrations. (A–C) Time-lapse images of dividing Wee1Co/−;TM-Cre MEFs either treated with ethanol (ctr) (A) or 4-hydroxytamoxifen (4-HT) (B, C). Representative images show either normal mitosis (A) or mitotic cells that cannot complete mitosis (B) or cells that withdraw from mitosis (C). (D) Summary of mitotic abnormalities in Wee1 mutant MEFs during live imaging. (E) Tamoxifen inducible Wee1 knockout MEFs either in the absence (a, b, c, d) or presence (e, f, g, h) of 4-HT were stained with antibodies against α-tubulin (red), Aurora-B (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (F) Quantification of the mitotic defects observed in Wee1-deficient cells after treatment with 4-HT. (G) Wee1 knockout MEFs either in the absence (a, b) or presence (c, d) of 4-HT were stained with pericentrin (red) and DAPI (blue).
Figure 2
Figure 2
WEE1 controls levels of cell-cycle regulators. (a) Western blots showing levels of Aurora-A, Aurora-B, Cyclin B1, Cdh1, securin and Plk1 in unsynchronized tamoxifen inducible Wee1 knockout MEFs. Cells were treated either with ethanol (ctr) or 4-hydroxytamoxifen (4-HT). (b) Western blot analysis in synchronized tamoxifen inducible Wee1 knockout MEFs after mitotic release. Cells were synchronized by serum starvation for 72 h, replaced with normal media containing nocodazole for 18 h, and then mitotic cells collected by shake off were replated and harvested at indicated time points for western blot analysis. (c) 293T cell were transfected with WEE1 shRNA or non-_targeting control shRNA for 24 h to collect whole-cell lysates for immunoblot analysis. (d) 293T cells were transfected with human WEE1 complementary DNA and levels of different cell-cycle proteins were checked by western blotting. (e) Wild-type (WT) and kinase-dead mutant (MT) WEE1 constructs were expressed in 293T cells and levels of cell-cycle proteins were examined by immunoblot. (f) Tamoxifen inducible Wee1 knockout MEFs were cultured in the presence of MG132 for the indicated time points and western blot was performed for Cyclin B1, securin, Plk1 and Aurora-B.
Figure 3
Figure 3
WEE1 interacts with APC/C. (a) Flag-WEE1 was immunoprecipitated and immunoblots of interacting proteins of the APC/C complex are shown. (b, c) Reciprocal immunoprecipitation of ectopically expressed Cdh1 (left) and Cdc20 (right) with WEE1 by using antibodies either against HA-tagged Cdh1 and Cdc20 (b) or flag-tagged WEE1 (c). (d, e) Co-immunoprecipitation of endogenous WEE1 with APC/C after immunoprecipitating endogenous Cdc27 in HeLa cells (d) as well as overexpressed Cdc20 and Cdh1 in 293T cells (e).
Figure 4
Figure 4
WEE1 negatively regulates APC/C activity. (a) APC/C was isolated from interphase Xenopus egg extracts and incubated in the absence (lane 1) or presence of recombinant human Cdh1 protein (lane 2), or APC/C was isolated from cell lysates from Wee1Co/−;TM-Cre MEFs treated either with ethanol (ctr) (lane 3) or 4-HT (lane 4). The ubiquitination activity of APC/C was assayed with a Myc-tagged N-terminal fragment of human cyclin B1. The reaction mixtures were separated on SDS–PAGE and blotted with the anti-Myc antibody. The positions of the cyclin B1 substrate and the cyclin B1-ubiquitin conjugates are labeled. (b) Control and WEE1 knocked-down (shWee1) HeLa cells were transfected with a myc-tagged Aurora-A construct as well as HA-ubiquitin. After treatment with MG132, Aurora-A was immunoprecipitated using myc-agarose beads and samples were blotted with an anti-HA antibody. Same experiment was performed in cells overexpressing Cdh1 to induce APC/Cdh1 activity (lanes 4 and 5). (c) After transfecting Myc-tagged Aurora-A and HA-ubiquitin, Aurora-A was immunoprecipitated from Wee1Co/−;TM-Cre MEFs either untreated or treated with 4-HT in the presence of MG132. Samples were blotted with an anti-HA antibody. Same experiment was performed in cells overexpressing Cdh1 to induce APC/Cdh1 activity (lanes 4 and 5). (d) DNA content of Wee1Co/−;TM-Cre MEFs treated either with ethanol or 4-HT for 48h. Percentage of cells with >4N DNA content is shown (P < 0.05). Characteristic image of a multi-nucleated cell is shown in upper panel.
Figure 5
Figure 5
Conditional deletion of Wee1 in mammary gland caused increased cellularity. (a) Whole-mount imaging of mammary glands from 6-month-old virgin WT (left), Wee1Co/+;MMTV-Cre (middle) and Wee1Co/Co;MMTV-Cre mice (right). Boxed areas are enlarged and placed underneath. (b–d) Hematoxylin and eosin stained sections (b), BrdU-positive cells (c) and TUNEL-positive nuclei (d) in 6-month-old virgin WT (left), Wee1Co/+;MMTV-Cre (middle) and Wee1Co/Co;MMTV-Cre mice (right). Boxed areas in c are enlarged areas showing BrdU-positive cells.
Figure 6
Figure 6
Wee1 deletion in mammary gland results in mis-coordination of the cell cycle. (a) Mammary gland sections of Wee1+/+;MMTV-Cre, Wee1Co/+;MMTV-Cre and Wee1Co/Co;MMTV-Cre mice were stained with anti-phosphorylated histone-H3 Ser10 (pH3; red) and anti-BrdU antibodies (green). Representative images from Wee1 mutant mammary glands are shown and arrows indicate positive stained cells. Nuclei were counterstained with DAPI. (b) Epithelial cells from tissue sections stained as described before were counted and the percentage of either BrdU (left) or phospho-H3 (right) positive cells is shown. (c) Wee1-defficiency in mammary gland causes DNA damage. Mammary glands from WT, Wee1Co/+;MMTV-Cre and Wee1Co/Co;MMTV-Cre mice were stained with an antibody against 53BP1. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and arrows indicate positive stained cells from mutant mammary glands. (d) Quantification of the percentage of mammary cells displaying positive staining for 53BP1. (e) Mammary glands from Wee1+/+;MMTV-Cre, Wee1Co/+;MMTV-Cre and Wee1Co/Co;MMTV-Cre mice were immunoblotted with antibodies against Aurora-B and securin. (f) Mammary glands from Wee1+/+;TM-Cre, Wee1Co/+;TM-Cre and Wee1Co/Co;TM-Cre mice either untreated or treaded with tamoxifen were immunoblotted with antibodies against Aurora-A and securin. (g) Liver (upper) and testis (lower) from same mice as described in (f) were checked for levels of mitotic regulators by immunoblot.
Figure 7
Figure 7
Tumor formation in mice carrying mammary-specific deletion of Wee1. (a) Kaplan–Meier survival curve of mice with different genotypes as indicated. (b) Representative hematoxylin and eosin stained (H&E) slides from mammary tumors from two Wee1Co/+;MMTV-Cre mice. (c) Sections from mammary tumors from Wee1Co/+;MMTV-Cre mice were stained with antibodies against HER2 and ER by immunohistochemistry. (d) Indirect immunofluorescence in mammary tumors from Wee1Co/+;MMTV-Cre mice with antibodies against SMA, CK14, CK18 and CK19. (e) Metaphases of cell lines developed from primary tumors showing double minute chromosomes (left) and polyploidy (middle). Quantification of chromosome numbers in Wee1-deficient tumor cells is shown (right).
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