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Article |
Loss of spindle assembly checkpoint–mediated inhibition of Cdc20 promotes tumorigenesis in mice
Correspondence to Pumin Zhang: pzhang{at}bcm.tmc.edu
Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and therefore aneuploidy by blocking premature separation of sister chromatids. Thus, SAC, much like the DNA damage checkpoint, is essential for genome stability. In this study, we report the generation and analysis of mice carrying a Cdc20 allele in which three residues critical for the interaction with Mad2 were mutated to alanine. The mutant Cdc20 protein (AAA-Cdc20) is no longer inhibited by Mad2 in response to SAC activation, leading to the dysfunction of SAC and aneuploidy. The dysfunction could not be rescued by the additional expression of another Cdc20 inhibitor, BubR1. Furthermore, we found that Cdc20AAA/AAA mice died at late gestation, but Cdc20+/AAA mice were viable. Importantly, Cdc20+/AAA mice developed spontaneous tumors at highly accelerated rates, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism.
© 2009 Li et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Before anaphase, sister chromatids are held together by cohesin complexes that resist the pulling force generated by the microtubule spindle. It is the dissolution of sister cohesion that allows anaphase to happen. The cohesin complexes are composed of protein subunits encoded by Smc1, Smc3, Scc1/Mcd1, and Scc3 and are thought to form a ring structure that encloses sister chromosomes (Nasmyth, 2005). At the onset of anaphase, the Scc1 subunit of the cohesin complex is cleaved by separase, a CD clan protease of the caspase family (Uhlmann et al., 2000), leading to the opening of the ring and release of sister chromatids. The timing of anaphase is controlled by spindle assembly checkpoint (SAC), an elaborate biochemical mechanism that ensures that sister chromatids are held together by cohesion rings until all of the chromosomes have achieved bivalent spindle attachments. By doing that, SAC prevents chromosome missegregation and aneuploidy. Dysfunctional SAC likely underlies the CIN phenotype observed in cancer cells.
SAC is activated when the kinetochores are not occupied by microtubules or when there is no tension at the kinetochores (Lew and Burke, 2003; Pinsky and Biggins, 2005). A single lagging chromosome is sufficient to activate SAC and cause an arrest in metaphase (Rieder et al., 1995). The same arrest is induced upon treating cells with spindle microtubule-disrupting agents such as nocodazole or colcemid. SAC activation (Diaz-Martinez and Yu, 2007) results in the inhibition of APC–Cdc20 by Mad2 and BubR1, and thus, the stabilization of securin and cyclin B1. Securin is an inhibitor of separase, and cyclin B1–Cdk1 kinase can phosphorylate separase (Stemmann et al., 2001). Phosphorylation of separase opens up the site for the binding and inhibition by the Cdk1–cyclin B1 complex (Gorr et al., 2005; Boos et al., 2008). Therefore, separase is dually inhibited by securin and phosphorylation when the checkpoint is activated, preventing premature separation of sister chromatids. These two inhibitory mechanisms are redundant in somatic cell lineages (Mei et al., 2001; Huang et al., 2005, 2008), but the phosphorylation is uniquely required in mouse embryonic germ cells (Huang et al., 2008). Furthermore, the stabilization of cyclin B1 prevents other events necessary for mitotic exit, leading to cell cycle arrest at prometaphase (Morgan, 1999).
Genetic analyses in budding yeasts have clearly demonstrated that the SAC is essential in preventing CIN (Li and Murray, 1991; Yamamoto et al., 1996). The discovery of mutations in BUBR1 and BUB1 in a subset of colon cancer cell lines (Cahill et al., 1998) suggests a weakened spindle checkpoint as the cause of CIN that contributed to the oncogenic process, which was further substantiated by the finding that BUBR1 is mutated in mosaic variegated aneuploidy, a rare human disorder characterized by increased percentage of aneuploid cells (usually >25%) and predisposition to childhood cancers (Hanks et al., 2004). To determine the role of spindle checkpoint in tumorigenesis, a large amount of efforts have gone to the generation and analysis of mice with targeted deletions in various SAC components. Because the spindle checkpoint is essential in mice, the analyses were restricted to heterozygous mice or mice carrying hypomorphic alleles. However, despite the compromises in the checkpoint, these mice did not display the expected large increases in the rate of spontaneous tumor development (Michel et al., 2001; Babu et al., 2003; Baker et al., 2004; Dai et al., 2004; Iwanaga et al., 2007). Furthermore, these spindle checkpoint components may have functions outside the checkpoint, and it might be the noncheckpoint-related functions that contribute to tumorigenesis when disrupted. Thus, whether the SAC is an as important tumor-suppressing mechanism as the DNA damage checkpoint is in question.
In this study, we report the generation and analysis of mice carrying a mutant Cdc20 allele (Cdc20AAA) in which the Mad2-binding sites in Cdc20 were mutated. The mutant Cdc20 protein can no longer be inhibited by Mad2 and renders the spindle checkpoint dysfunctional. These mice contain a high percentage of aneuploid cells and develop spontaneous neoplasms at a much increased rate, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism. Furthermore, analysis of the mutant cells revealed that the timing of anaphase depended on Mad2–Cdc20 interaction, and additional expression of BubR1 could not rescue the spindle checkpoint defects caused by AAA-Cdc20.
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We derived mouse embryonic fibroblasts (MEFs) from E12.5 embryos. Cdc20AAA/AAA MEFs grew noticeably slower than the wild-type control even at early passages (<3; Fig. 3 A), and the growth succumbed to a complete stop as a result of massive cell death as passage number increased (Fig. 3 B). However, the culture could be rescued with immortalization by the expression of SV40 large T antigen, suggesting that the cell death was mediated by the p53, Rb, or both pathways. When immortalized MEFs (iMEFs) were treated with nocodazole, wild-type cells accumulated in mitosis, but Cdc20AAA/AAA cells failed to do so (Fig. 3 C), indicating that the mutation disrupts SAC in MEFs as well. Similar to the ES cells, Cdc20AAA/AAA iMEFs did not respond to taxol treatment (Fig. 3 D). Cdc20+/AAA iMEFs displayed a milder defect in the checkpoint function than the homozygous mutant cells (Fig. 3 C).
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Next, we tested whether overexpression of BubR1 could compensate for the lost interaction between Mad2 and Cdc20 in the mutant cells. Empty vector or BubR1-expressing vector together with a GFP-expressing plasmid were transfected into iMEFs. Mitotic indices of the transfected (GFP positive) cells were determined before and after 12-h nocodazole treatment. Although additional expression of BubR1 (Fig. 4 C) significantly increased the mitotic indices of wild-type cells even in the asynchronously growing population, it had little impact on the homozygous mutant cells either treated with nocodazole or untreated (Fig. 4 D), suggesting that the inhibition of Cdc20 by BubR1 requires the function of Mad2. In fact, the endogenous level of BubR1 was already noticeably elevated in Cdc20AAA/AAA cells (Fig. 4, A and C), but the SAC was still ineffective. It is unclear at the moment what causes the elevation in BubR1 levels in AAA-Cdc20 homozygous cells. In the heterozygous cells, additional BubR1 expression was able to improve the checkpoint function to some extent (Fig. 4 D), most likely working through the wild-type Cdc20 protein.
Abnormal mitoses in MEFs harboring Cdc20AAA
During normal mitotic divisions, SAC is essential in delaying anaphase until all sister chromatids are aligned at metaphase plate. We analyzed mitoses in asynchronously growing MEFs undisturbed by microtubule-disrupting agents. Microscopic observation indicated that the mitoses were highly abnormal in the mutants (Fig. 5, A and B). In metaphase (when a metaphase plate was clearly visible), the chromosomes in mutant cells were misaligned (Fig. 5 A), and in anaphase, there were lagging chromosomes or chromosome bridges in the mutant cells (Fig. 5 A). Quantitation of the abnormal mitoses are shown in Fig. 5 B. The abnormal mitoses in the mutant MEFs suggested that these cells should become aneuploid. Indeed, as early as passage 3, both heterozygous and homozygous mutant MEFs showed high percentages of cells with abnormal karyotypes, reaching 28% and 52%, respectively, whereas the wild-type cells only showed 5% aneuploidy (Fig. 5 C).
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8–10 min earlier than the control (Fig. 6 B), indicating that Mad2-mediated inhibition of Cdc20 is critical in the timing of anaphase. In agreement with the advancement of anaphase in mutant cells, Cdc20AAA/AAA MEFs contained reduced levels of securin and cyclin B1 (Fig. S2).
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35% aneuploid cells, whereas the wild type contained only 6%. To determine whether such a high percentage of aneuploidy has any impact on spontaneous tumorigenesis, we subjected cohorts of wild-type and Cdc20+/AAA mice to long-term (24 mo) observation of tumor development. These mice were under a mixed genetic background of C57BL/6 and 129SV. As shown in Fig. 7 B, Cdc20+/AAA mice had a significantly increased rate of tumor formation. Palpable tumors could be detected as early as 6.8 mo in the mutant mice. By 24 mo, 50% of the mutant mice developed tumors, whereas only 10% of the wild-type animals did. 38% of the mice that had tumors developed tumors at multiple organ sites. Fig. 7 C shows a couple of large images of the tumors. Logrank test indicated that the tumor-free curves of wild-type and mutant mice were significantly different with P < 0.0001. Among 29 tumor samples analyzed histopathologically, four were hepatomas (13.8%) and the rest were lymphomas. Spectrum karyotyping (SKY) analysis of one lymphoma sample demonstrated aneuploidy (2 N + 3) in this tumor (Fig. 7 D). The tumors developed in wild-type animals were all lymphomas.
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A complete lack of SAC is incompatible with viability in higher eukaryotes. Mad2-deficient mice show early embryonic lethality around the blastocyst stage (Dobles et al., 2000). Loss of Mad2 in Caenorhabditis elegans is also incompatible with the viability (Kitagawa and Rose, 1999). Other central components of the SAC, Bub1, BubR1, and Mad1, are all essential as well (Basu et al., 1999; Kitagawa and Rose, 1999; Babu et al., 2003; Wang et al., 2004). Although Mad2-deficient cells are nonviable (Michel et al., 2001), the nonviability could be rescued with the deletion of p53 (Burds et al., 2005). Thus, a complete lack of SAC function does not cause cell lethality by itself. Rather, the cells are eliminated by other protecting mechanisms such as p53-induced apoptosis, perhaps because of the severe aneuploid nature of these cells. However, the SAC is not "all or none." It can be compromised to certain degrees as indicated by the fact that the mice heterozygous for the essential components are viable despite clear defects in the checkpoint; e.g., not only do the cells derived from these mice arrest less efficiently than wild-type cells in response to microtubule disruption, there were also certain percentages of aneuploid cells present in the adult animals. The percentage of the aneuploid cells could be tolerated as high as >30% (BubR1 hypomorphic mice and our AAA-Cdc20 heterozygous mice). It is unclear at present whether such a high percentage of aneuploidy has any impact on normal physiology of the animals.
Despite the aneuploidy displayed by SAC mutants, only small increases in cancer susceptibility have been reported. For example, tumor incidence was increased (to 6%) in mice with severely reduced BubR1 levels (Baker et al., 2004). Also, 28% of mice heterozygous for Mad2 develop small, self-limiting, late onset (18–19 mo) papillary lung adenocarcinomas (Michel et al., 2001), and mice heterozygous for functional BubR1 or Bub3 are more prone to the development of colorectal (Dai et al., 2004) or lung (Babu et al., 2003; Dai et al., 2004) tumors after treatment with azoxymethane or DMBA (9,10-dimethyl-1,2-benzanthracene), respectively. More recently, mice heterozygous for Mad1 were reported to display a small increase (from 9 to 19%) in the incidence of spontaneous tumors at old ages (Iwanaga et al., 2007), and mice heterozygous for centromere protein E similarly displayed a small increase in tumor incidence (Weaver et al., 2007). These minor tumor phenotypes put the significance of the SAC in preventing the tumorigenesis in question. However, results from Cdc20+/AAA (this study) and Bub1-deficient mice (Jeganathan et al., 2007) suggest that the loss of SAC function can be highly tumorigenic. What could be the reason for the differences in the rates of tumor development among various mouse strains with SAC defects? One obvious possibility is the severity of the checkpoint defects and thus the rates of aneuploidy. Indeed, both Cdc20+/AAA and Bub1 hypomorphic mice contain high percentages of aneuploid cells (>30% when splenocytes were analyzed). However, mice deficient in BubR1 or doubly heterozygous for Bub3 and Rae1 contain a similarly high percentage of aneuploid cells, and yet, these mice are not tumor prone (Baker et al., 2004, 2006). One might argue that the premature aging process in BubR1-deficient and Bub3+/– Rae1+/– mice prevented these animals from developing tumors. However, such an argument could not be applied to centromere protein E+/– mice, which also display similarly high percentages of aneuploid cells as Cdc20+/AAA mice but only have a mild increase in tumor incidence (Weaver et al., 2007). Thus, the rate of aneuploidy cannot be the only determinant on the likelihood of tumorigenesis in SAC mutants. Another possibility is that Cdc20 and Bub1 may have functions outside mitosis. Loss (in the case of Bub1) or gain (in the case of AAA-Cdc20) of these functions in combination with aneuploidy may therefore drive the robust tumor development in these mice and potentially explain the difference in the tumor spectra between these two strains. Indeed, Cdc20 was suggested to function in regulating gene expression in mammalian cells (Yoon et al., 2004), and budding yeast CDC20 was found to be able to override G2/M DNA damage checkpoint when overexpressed (Lim and Surana, 1996) and needs to be repressed in S phase to prevent premature mitotic entry (Clarke et al., 2003). Given the fact that AAA-Cdc20 is no longer inhibited by Mad2, this mutant Cdc20 protein might have gained additional functions that help oncogenic transformation. In the case of Bub1, it seems to be required for the induction of cell death when chromosome missegregates (Jeganathan et al., 2007) and may have other unspecified functions that prevent tumorigenesis.
Both Mad2 and BubR1 can bind to Cdc20 directly. The Mad2-binding sites in Cdc20 are conserved in other Mad2-interacting proteins such as Mad1 (Zhang and Lees, 2001; Luo et al., 2002). However, the BubR1-interacting domain in Cdc20 remains ill defined (Luo et al., 2002). Recent studies in budding yeasts suggested that MAD3 (BubR1 in mammals) uses its KEN box to interact with CDC20 and inhibits APCCdc20 as a pseudosubstrate (Burton and Solomon, 2007), and it seems that a similar mechanism is used by BubR1 in mammals (Malureanu et al., 2009). One possibility for why both Mad2 and BubR1 are essential SAC components is stoichiometry. In other words, the number of Mad2 or BubR1 molecules is perhaps by themselves insufficient to inhibit all APCCdc20 complexes. However, our results (Fig. 4 E) indicate that this is unlikely the case. It is more likely that the mitotic checkpoint complex (MCC) containing Mad2, BubR1, Bub3, and Cdc20 blocks the E3 activity of APCCdc20 (Sudakin et al., 2001) instead of Mad2 and BubR1 acting separately. Indeed, MCC was much more potent in inhibiting APCCdc20 than Mad2 alone or BubR1 in complex with Bub3 (Sudakin et al., 2001), suggesting a synergy between Mad2 and BubR1 in enforcing the checkpoint. Recent biochemical and genetic complementation analyses suggest that MCC might be transient (Kulukian et al., 2009; Malureanu et al., 2009). The interaction between Mad2 and Cdc20 is to make Cdc20 susceptible to BubR1 binding and inhibition. In other words, activated Mad2 acts as a catalyst for the formation of BubR1–Bub3–Cdc20 or BubR1–Bub3–APCCdc20 complexes. Therefore, Mad2 deletion, BubR1 deletion, or loss of Mad2–Cdc20 interaction all lead to active APCCdc20 and thus no SAC. However, Cdc20AAA is certainly less disruptive than Mad2 or BubR1 deletion because Cdc20AAA/AAA mice could survive up to midgestation, whereas Mad2- and BubR1-deficient mice died much earlier. This discrepancy is likely caused by the incomplete disruption of the Mad2–Cdc20 interaction by the mutation. There might still be some residual interaction between Mad2 and the mutant Cdc20 that could not be detected with coimmunoprecipitation.
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Isolation and analysis of MEFs
MEFs were isolated from 12.5-d-postcoitum embryos and cultured in DME supplemented with 2 mM glutamine, 1% penicillin/streptomycin, and 15% FBS. Immortalization of MEF cells were realized via retroviral expression of SV40 large T antigen. For live image analysis, MEF cells (passage 2) were infected with retroviruses carrying H2B-YFP (provided by J. van Deursen, Mayo Clinic, Rochester, MN) and were recovered for 24 h after the infection. The cell culture dish containing the infected cells was placed on the temperature-controlled warm stage of a microscope (Axiovert 200; Carl Zeiss, Inc.) equipped with an environmental chamber. The temperature was maintained at 37°C and the CO2 level at 10% with the CTI3700 controller (Carl Zeiss, Inc.). The cells were imaged every 5 min for 4 h. Imaging software (WS/20A; Carl Zeiss, Inc.) was used to analyze the progression of mitosis. At least 30 mitotic cells were analyzed. To express BubR1, iMEFs were transfected with pCDNA3-BubR1 together with a GFP-expressing plasmid at a 4:1 ratio. 24 h after the transfection, the cells were treated with nocodazole for 8 h, fixed with 4% PFA, and stained with DAPI. The number of mitotic cells was counted in GFP-positive populations.
For Western blotting, the cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail [Roche]). 50 µg total protein was separated in SDS-PAGE and immunoblotted with antibodies against cyclin B1 (Santa Cruz Biotechnology, Inc.), securin (NeoMarkers), or Cdc20 (Santa Cruz Biotechnology, Inc. and Millipore).
For coimmunoprecipitation, the iMEFs were lysed with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40), and the lysates were incubated with anti-Cdc20 antibody (Millipore) or anti-Flag antibodies (Sigma-Aldrich) for 90 min at 4°C. The immune complexes were precipitated with protein G agarose beads (GE Healthcare), washed four times with NETN buffer, and eluted with 2x SDS loading buffer. The eluted proteins were separated by SDS-PAGE and probed with antibodies against Cdc20, BurbR1, and Mad2 (Santa Cruz Biotechnology, Inc.).
Karyotype analysis
To isolate splenocytes, spleens were collected and minced between two microscope slides. The released the cells were cultured for 48 h in RPMI 1640 supplemented with 10% FBS, 5 µg/ml lipopolysaccharide, 1 µg/ml anti–mouse CD28, and 1 µg/ml anti–mouse CD3e (BD).
To prepare chromosome spreads, MEFs (at passage 3) and splenocytes were treated with 10 µM MG132 (Calchemico) for 5 h at 37°C, harvested and resuspended in 5 ml 0.075 M KCl, and incubated in the hypotonic solution at 37°C for 10 min. The cells were fixed in Carnoy's solution (methanol/acetic acid [3:1]), washed with PBS, and resuspended in 0.5 ml Carnoy's solution. The cell suspension was dropped onto prewetted microscope slides and air dried. Chromosomes were visualized by 10-min staining in 5% Giemsa solution.
For SKY, lymphoma cells were isolated and cultured. Chromosome spreads were prepared from asynchronously growing population of cells and stained with chromosome-specific probes in our cytogenetic core laboratory.
Histology analysis
Embryos or tumor tissues were fixed overnight in 4% PFA/PBS, pH 7.4, and embedded in paraffin. 4-mm sections were prepared and stained with hematoxylin and eosin according to standard protocols. For immunostaining of activated caspase 3, tissue sections were boiled for 10 min in citrate buffer (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) in a microwave oven to retrieve antigens and were stained with antiactive caspase 3 antibodies (Cell Signaling Technology).
Imaging
For regular fluorescence imaging, we used a microscope (E800; Nikon) with a Plan Fluor 100x/1.30 oil objective (Nikon). Images were captured with a digital camera (SPOT-RT model 2.3.1; Diagnostic Instruments, Inc.). Fluochromes used are DAPI, Texas red, and FITC. For live imaging of YFP-labeled cells, we used a microscope (Axiovert 200; Carl Zeiss, Inc.) with a Plan Fluor 20x/0.30 objective (Carl Zeiss, Inc.). Images were taken with a digital camera (AxioCam; Carl Zeiss, Inc.).
Online supplemental material
Fig. S1 shows the embryonic phenotype of Cdc20AAA/AAA mice, and Fig. S2 shows immunostaining of securin and cyclin B1 in the mutant MEF cells in mitosis. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200904020/DC1.
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Acknowledgments |
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M. Li is supported by a postdoctoral training grant from the National Institutes of Health. This work was funded by research grants from the National Cancer Institute (CA122623 and CA116097 to P. Zhang).
Submitted: 3 April 2009
Accepted: 18 May 2009
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