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Cyclin B2 and p53 control proper timing of centrosome separation

Nature Cell Biology volume 16, pages 535–546 (2014)Cite this article

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A Corrigendum to this article was published on 01 October 2014

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Abstract

Cyclins B1 and B2 are frequently elevated in human cancers and are associated with tumour aggressiveness and poor clinical outcome; however, whether and how B-type cyclins drive tumorigenesis is unknown. Here we show that cyclin B1 and B2 transgenic mice are highly prone to tumours, including tumour types where B-type cyclins serve as prognosticators. Cyclins B1 and B2 both induce aneuploidy when overexpressed but through distinct mechanisms, with cyclin B1 inhibiting separase activation, leading to anaphase bridges, and cyclin B2 triggering aurora-A-mediated Plk1 hyperactivation, resulting in accelerated centrosome separation and lagging chromosomes. Complementary experiments revealed that cyclin B2 and p53 act antagonistically to control aurora-A-mediated centrosome splitting and accurate chromosome segregation in normal cells. These data demonstrate a causative link between B-type cyclin overexpression and tumour pathophysiology, and uncover previously unknown functions of cyclin B2 and p53 in centrosome separation that may be perturbed in many human cancers.

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Figure 1: Ccnb1 and Ccnb2 transgene production and expression analyses.
Figure 2: B-type cyclins induce distinct chromosome missegregation when overexpressed.
Figure 3: Cyclin B2 overexpression causes aurora-A-mediated hyperactivation of Plk1.
Figure 4: Endogenous cyclin B2 regulates aurora-A-mediated centrosome separation.
Figure 5: Cyclin B2 and p53 antagonistically control aurora-A-mediated centrosome separation.
Figure 6: Cyclins B1 and B2 drive tumorigenesis when overexpressed.

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  • 09 September 2014

    In the version of this Article originally published, the authors incorrectly cited ref. 39, and this has now been deleted from the online versions of the Article. In addition, the authors omitted four key references, which have now been added as below: In the following sentences on page 540: "Plk1 activity typically increases in late G2, resulting in phosphorylation and activation of Mst2, which in turn phosphorylates and activates Nek2A67,68. Activated Nek2A then phosphorylates C-Nap1 and rootletin, two proteins that hold duplicated centrosomes, resulting in centrosome separation69,70". In the legend of Fig. 5j, as well as an additional citation of ref. 38: "Cyclin B2–Cdk1, on the other hand, triggers the phosphorylation of the residual aurora A protein pool in a timely manner to induce phosphorylation of centrosome-associated Plk1 and permit centrosome disjunction38,67–70". Additional references: 67. Mardin, B. R. et al. Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction. Nat. Cell Biol. 12, 1166–1176 (2010). 68. Mardin, B. R., Agircan, F. G., Lange, C. & Schiebel, E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction. Curr. Biol. 21, 1145–1151 (2011). 69. Fry, A. M.et al. C Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J. Cell Biol. 141, 1563–1574 (1998). 70. Bahe, S., Stierhof, Y. D., Wilkinson, C. J., Leiss, F. & Nigg, E. A. Rootletin forms centriole-associated filaments and functions in centrosome cohesion. J. Cell Biol. 171, 27–33 (2005).

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Acknowledgements

We thank W. Zhou and M. Li of Mayo Clinic’s Gene Knockout Mouse Core Facility for ES cell microinjection and chimaera breeding, and L. Malureanu, K. Jeganathan and D. Baker for help with live-cell imaging, karyotyping and tumour analyses, respectively. We are grateful to P. Galardy, R. Ricke, R. Naylor, D. Baker and L. Malureanu for discussions and critical evaluation of this manuscript, J. Salisbury for sharing antibody, J-M. Peters for sharing the Scc1 construct, D. Compton and L. Kabeche for sharing photoactivable GFP–α-tubulin construct and help with measurements of microtubule dynamics, V. Shridhar for human lung (tumour) samples and the Mayo Clinic Cytogenetics shared resource for FISH analysis. This work was supported by the National Institutes of Health (CA126828 to J.M.v.D.).

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  1. Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    Hyun-Ja Nam & Jan M. van Deursen

  2. Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

    Jan M. van Deursen

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H-J.N. and J.M.v.D. conceived the research and designed experiments. H-J.N. carried out experiments and analysed results. H-J.N. and J.M.v.D. discussed results and wrote the paper.

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Correspondence to Jan M. van Deursen.

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Integrated supplementary information

Supplementary Figure 1 Analysis of CcnB1 and CcnB2 transgene expression.

(a and b) Western blot analysis of tissue extracts from 6-week-old dox-treated HA-CcnB1 (strains T7 and T11), HA-CcnB2 (strains T15 and T16) and control (TA) transgenic mice. Ponceau S staining of blotted proteins served as a loading control (c) Immunoblots of tissue extracts from 6-week-old dox-treated and untreated HA-CcnB1T7 or HA-CcnB2T16 transgenic mice demonstrating that transgene expression is tightly controlled by dox. All blots are representative of two independent experiments.

Supplementary Figure 2 Cyclin B1 protein levels and Cdk substrate phosphorylation are increased in CcnB1 transgenic MEFs during mitosis.

(a) Dox-treated and untreated CcnB1T7 MEFs at various stages of cell cycle progression stained for cyclin B1. P-H3S10 staining was used for cell cycle staging. (b) Quantification of cyclin B1 signals of dox-treated and untreated CcnB1T7 MEFs at various stages of the cell cycle. (c) As in a but stained for phosphorylated Cdk substrates. γ-Tubulin staining was used for cell cycle staging. (d) Quantification of pCdk substrate signals of dox-treated and untreated CcnB1T7 MEFs at various stages of the cell cycle. DNA was visualized with Hoechst. Scale bars, 10 μm. Data in b and d represent mean ± s.e.m. (per group, we analysed ≥10 cells in b, and ≥8 cells in d). Statistical significance was determined by a two-tailed, unpaired t-test. Statistics source data are provided in Supplementary Table 2. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 3 Cyclin B2 protein levels and Cdk substrate phosphorylation are increased in CcnB2 transgenic MEFs throughout the cell cycle.

(a) Dox-treated and untreated CcnB2T16 MEFs at various stages of cell cycle progression stained for cyclin B2. γ-Tubulin staining was used for cell cycle staging. (b) Quantification of cyclin B2 signals of dox-treated and untreated CcnB2T16 MEFs at various stages of the cell cycle. (c) As in a but stained for phosphorylated Cdk substrates. γ-Tubulin staining was used for cell cycle staging. (d) Quantification of pCdk substrate signals of dox-treated and untreated CcnB2T16 MEFs at various stages of the cell cycle. DNA was visualized with Hoechst. Scale bars, 10 μm. Data in b and d represent mean ± s.e.m. (per group, we analysed ≥8 cells in b and d). Statistical significance was determined by two-tailed, unpaired t-test. Statistics source data are provided in Supplementary Table 2. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Figure 4 Cyclin B1 overexpression does not appear to disrupt early mitosis.

(a) Quantification of total cyclin B1 signal in dox-treated CcnB1T7 anaphases with and without chromatin bridges. Data represent mean ± s.e.m. Three independent MEF lines were included in the analysis. (b) Time-lapse images of biosensor-positive CcnB2T16 MEFs progressing through mitosis in the absence or presence of transgenic cyclin B2. Bars, 5 μm. (c) Quantification of biosensor fluorescence at chromosomes as cells progress through mitosis. Data represent mean ± s.e.m. from five independent experiments. (d) Western blot analysis of lysates from induced and non-induced CcnB1T7 MEFs for Top2a (blots are representative of two independent experiments). (e) Images of CcnB1T7 prometaphases immunostained for centromeres and Top2a. Insets show enlarged centromeres from boxed region. Bar, 5 μm. (f and g) Quantitation of RPA2- or γH2AX/53BP1-positive foci in induced and non-induced CcnB1T7 and CcnB2T16 MEFs. Data represent mean ± s.e.m. from three independent MEF lines (we analysed ≥100 cells per line). (h) Timing of early stages of mitosis of induced and non-induced CcnB1T7 MEFs by live-cell imaging. P, prophase; PM, prometaphase; M, metaphase; and A, anaphase onset. Data represent mean ± s.e.m. from three independent MEF lines (we analysed 10 cells per line) (i) Analysis of S/G2 duration in induced and non-induced CcnB1T7 MEFs using FUCCI technology. Data represent mean ± s.e.m. from three independent MEF lines (we analysed ≥10 cells per line). (j) Growth curves of induced and non-induced CcnB1T7 and CcnB2T16 MEFs (P3). Data represent mean ± s.e.m. from three independent MEF lines. *P < 0.05 denotes a statically significant cell number at day 5. Statistical significance was determined by a two-tailed unpaired t-test. Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 5 Characterization of the chromosome lagging phenotype of cyclin B2 overexpressing cells.

(a) Nocodazole- and taxol-challenge assays on CcnB1T7 and CcnB2T16 MEFs with or without transgene induction. Data represent mean ± s.e.m. from three independent MEF lines (we analysed ≥10 cells per line). (b) Quantification of cells with misaligned chromosomes following release from monastrol and MG132 exposure. Data represent mean ± s.e.m. from three independent MEF lines (we analysed ≥25 cells per line). (c) Measurement of kinetochore-microtubule dynamics in cyclin B2 transgenic MEFs. Normalized fluorescence intensity over time after photoactivation of spindles in prometaphase cells. Data represent mean ± s.e.m. of 8 cells per genotype. (d and e) Images of methanol-fixed CcnB2T16 MEFs immunostained for the centrosome marker Centrin 2 and either cyclin B2 (d) or HA (e). DNA was visualized with Hoechst. Scale bar, 10 μm. (f) Dox-treated CcnB2T16 MEFs in G2 analysed for centrosome distance and level of HA cyclin B2 expression (29 cells were analysed). Note that there is a correlation between level of overexpression and centrosome distance. (g) Accelerated centrosome separation at various concentrations of Plk1 inhibitor. Data represent mean ± s.e.m. from three independent MEF lines (we analysed 20 cells per line). Cells with centrosome distances ≤8 μm were classified as accelerated. (h) Quantitation of p-Aurora-A signals at centrosomes at the various stages of mitosis. Data represent mean ± s.e.m. of 7–11 cells per group. (i) Quantitation of p-Plk1T210 signal at centrosomes in G2 CcnB2T16 MEFs (a.u., arbitrary unit). Data represent mean ± s.e.m. from three independent MEF lines (we analysed 10 cells per line). (j) Incidence of spindle geometry abnormalities at various concentrations of Aurora A inhibitor MLN8054. Data represent mean ± s.e.m. from three independent MEF lines (we analysed 20 cells per line). (k) Images of CcnB2T16 prometaphases immunostained for p-Plk1T210, p-BubR1T680, centromeres. p-BubR1T680 phosphorylation by p-Plk1T210 mediates recruitment of PP2A to kinetochores to prevent uncontrolled Aurora B activity and destabilization of microtubule-kinetochore attachments. DNA was visualized with Hoechst. Bar, 10 μm. (l) Quantification of p-BubR1T680 signal in presence or absence of elevated cyclin B2. Note that prometaphases accumulate normal levels of p-BubR1T680, indicating that attachment error correction at kinetochores is unperturbed when cyclin B2 levels are upregulated. Data represent mean of 10 cells per group. (m) Spindle position measurements. Data represent mean ± s.d. of 20 cells per group. Statistical significance for was determined by a two-tailed, unpaired t-test. Statistics source data are provided in Supplementary Table 2.

Supplementary Figure 6 p53 loss increases Aurora A and Plk1 transcript and protein levels.

(a) Western blots of extracts from wild-type and p53−/− MEFs. Actin probe and Ponceau S staining served as loading controls (blots are representative of two independent experiments). (b) Aurka and Plk1 transcript levels in wild-type and p53/MEFs analysed by qRT-PCR. Data represent mean ± s.e.m. from three independent experiments (we measured duplicate of two independent MEF lines per experiment). GAPDH transcript levels were used for normalization. Values were normalized against wild-type values. Consistent with evidence that p53 mediates transcriptional repression of Plk166, we find that Plk1 is upregulated in p53−/− MEFs. However, it is important to emphasize that even though Plk1 expression is upregulated, Plk1 hyperactivity in p53−/− MEFs remains fully dependent on Aurora A hyperactivity as low amounts of Aurora A kinase MLN8054 inhibitor restore proper timing of centrosome separation, spindle geometry, and accurate chromosome segregation (Fig. 5g–i). (c) Quantitation of p-Aurora A intensity at centrosomes in G2 cells. (d) Quantitation of p-Plk1T210 intensity at centrosomes G2 cells. Data in c and d represent mean ± s.e.m. from three independent MEF lines (we analysed 10 cells per line). Statistical significance was determined by a two-tailed, unpaired t-test. Statistics source data are provided in Supplementary Table 2. *P < 0.05, ***P < 0.001.

Supplementary Figure 7 CCNB1 and CCNB2 are frequently overexpressed in human lung carcinomas.

CCNB1 (a), CCNB2 (b), Ki67 (c) and BUB1 (d) transcript levels in normal human lung tissue, lung adenocarcinomas, and squamous cell lung carcinomas analysed by qRT-PCR. Data represent the mean of duplicate measurements. TBP transcript levels were used for normalization. The relative fold CCNB1, CCNB2, Ki67 or BUB1 transcript in tumors was normalized to the average of the normal lung tissue values. Ki67 and BUB1 transcripts were used as proliferation and mitotic markers, respectively.

Supplementary Figure 8 Quantitation of small intestinal tumors and colon tumor histopathology 30 day after dox withdrawal.

(a) Tumor multiplicity in small intestine. n represents the number of mice analysed. Data represent mean ± s.e.m. Statistical significance was determined by two-tailed unpaired t-test. (b) Hematoxylin-eosin-stained sections of colonic polyps of mice that were taken off dox for the last 30 days of the experiment. Black and red scale bars are 2 mm and 100 μm, respectively.

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Nam, HJ., van Deursen, J. Cyclin B2 and p53 control proper timing of centrosome separation. Nat Cell Biol 16, 535–546 (2014). https://doi.org/10.1038/ncb2952

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