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Oncogene-like induction of cellular invasion from centrosome amplification

Nature volume 510, pages 167–171 (2014)Cite this article

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Abstract

Centrosome amplification has long been recognized as a feature of human tumours; however, its role in tumorigenesis remains unclear1. Centrosome amplification is poorly tolerated by non-transformed cells and, in the absence of selection, extra centrosomes are spontaneously lost2. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumours3, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumour progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behaviour is similar to that induced by overexpression of the breast cancer oncogene ERBB2 (ref. 4) and indeed enhances invasiveness triggered by ERBB2. Our data indicate that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell–cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation.

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Figure 1: Invasive behaviour of epithelial cells triggered by centrosome amplification.
Figure 2: The induction of aneuploidy does not generate invasive acini.
Figure 3: Centrosome amplification disrupts normal cell–cell adhesion because of Rac1 activation.
Figure 4: Effects of centrosome amplification are mediated by increased nucleation of centrosomal microtubules.

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Primary accessions

Gene Expression Omnibus

Data deposits

The SNP data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through accession number GSE55042.

Change history

  • 02 May 2014

    In Fig. 4g, the plus and minus symbols were incorrect; these were fixed on 2 May 2014.

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Acknowledgements

We are grateful to S. Jhaveri-Schneider, S. Muthuswamy, M. Bettencourt-Dias, R. Basto, D. Calado, N. Ganem, A. Spektor, M. Kwon and B. Atkins for comments or discussion of the manuscript; the Nikon Imaging Center at Harvard Medical School; J. Waters, J. Rosenberg and H. Elliott from the Image and Data Analysis Core at Harvard Medical School for their help with FRET microscopy and analysis; L. Cameron of the Confocal and Light Microcopy Facility at Dana-Farber Cancer Institute; H. Li of SNP analysis; A. Bui for help establishing the 3D cultures; Q. Tseng for establishing MCF10A doublets on micropatterns; and C. Sproat and J. Marshall for help with the organotypic cultures. Reagents were kindly provided by L. Pelletier, J. Locarek, M. Matsuda, A. Hall and J. Marshall. S.A.G. is supported by an FCT grant HMSP-CT/SAU-ICT/0075/2009. M.T. is supported by the ERC grant 310472 and the ISI program of BPIFrance. D.P. is an HHMI investigator and is supported by an NIH grant GM083299-1.

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Author notes

  1. Susana A. Godinho & Cheuk T. Leung

    Present address: Present addresses: Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK (S.A.G.); Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, USA (C.T.L.).,

Authors and Affiliations

  1. Department of Pediatric Oncology, Howard Hughes Medical Institute, Dana-Farber Cancer Institute and Pediatric Hematology/Oncology, Children’s Hospital, Boston, 02115, Massachusetts, USA

    Susana A. Godinho, Remigio Picone, Regina Dagher & David Pellman

  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Susana A. Godinho, Remigio Picone, Regina Dagher, Cheuk T. Leung, Joan S. Brugge & David Pellman

  3. Institut de Recherche en Technologie et Science pour le Vivant, UMR5168 CEA/UJF/INRA/CNRS, Grenoble, France,

    Mithila Burute & Manuel Théry

  4. Hôpital Saint Louis, Institut Universitaire d’Hematologie, U1160 INSERM/AP-HP/Université Paris Diderot, Paris 75010, France,

    Mithila Burute & Manuel Théry

  5. CYTOO SA, Grenoble 38054, France,

    Mithila Burute

  6. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Ying Su & Kornelia Polyak

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Contributions

S.A.G. and D.P. designed the experiments and wrote the manuscript. S.A.G. conceived, conducted and performed data analysis for most experiments. R.P. conceived and conducted all FRET experiments and Fig. 3a. M.T. and M.B. contributed with micro-pattern fabrication, Fig. 3d, Extended Data Fig. 6e, d and Extended Data Fig. 8c–e. R.D. contributed with Fig. 2b and Extended Data Fig. 5a. Y.S., K.P., C.T.L. and J.S.B. provided assistance with 3D cultures. All authors contributed with discussions and edited the manuscript.

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Correspondence to Susana A. Godinho or David Pellman.

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Extended data figures and tables

Extended Data Figure 1 Characterization of cells after transient overexpression of PLK4 or PLK41–608.

a, Scheme of the experimental design to induce centrosome amplification. Transient overexpression of PLK4 and PLK41–608 was achieved by addition of Dox for 48 h (2D culture) followed by removal of Dox and growth in 3D culture for 4 days in the absence of Dox. b, qRT–PCR showing the levels of induction of the PLK41–608 and wild-type PLK4 transgenes in cells after 48 h of Dox. Error bars represent mean ± s.e. from 3 independent experiments. c, qRT–PCR showing the expression of PLK4 after 48 h of Dox (2D) and after 4 days in 3D cultures (3D). Note that PLK4 overexpression after 4 days in 3D cultures is down to control levels after Dox removal. Error bars represent mean ± s.e. from 3 independent experiments. d, Proliferation curve of cells after induction of PLK4 and PLK41–608 over 72 h. Centrosome amplification decreases cell proliferation. Error bars represent mean ± s.e. from 3 independent experiments. e, The fraction of cells with centrosome amplification at the indicated time points after PLK4 induction. Note that, because centrosome number is quantified in mitotic cells, this result demonstrates that cells with extra centrosomes can enter mitosis even after 72 h of Dox treatment. Error bars represent mean ± s.e. from 3 independent experiments. f, Fraction of cells with centrosome amplification in an independently generated MCF10A.PLK4 cell line. Error bars represent mean ± s.e. from 3 independent experiments. g, Corresponding fraction of invasive acini in 3D cultures. Error bars represent mean ± s.e. from 3 independent experiments. h, Centrosome amplification (PLK4 overexpression, +Dox) in non-transformed keratinocytes (HaCaTs) promotes invasion in the organotypic culture model. Images show H&E staining of sections of HaCaTs cells. Black arrows indicate cells invading the matrix. Note that the invasion of groups of cells was only detected in the +Dox condition (black arrowhead). Scale bar, 100 μm. Graph shows quantification of the percentage of cells that invade. Each dot in the graphic represents the percentage of invasion in each individual well per experiment analysed. The P value was derived from unpaired two-tailed Student’s t-test *P < 0.05). i, Collagen-I degradation induced by centrosome amplification (green). Collagen degradation is visualized by DQ-Col-I, which becomes fluorescent after degradation because of fluorophore dequenching. Scale bar, 10 μm. See Methods for more details.

Extended Data Figure 2 Characterization of evolved diploid and tetraploid cells.

a, Scheme of the experimental design to obtain fresh MCF10A tetraploid cells with extra centrosomes (4N) and ‘evolved’ tetraploid cells that lost the extra centrosomes (4N.evo), as previously described2. b, FACS profiles of ‘evolved’ diploid (2N.evo) and tetraploid cells (4N.evo). c, Western blotting to detect E-cadherin in the ‘evolved’ cells indicates that 4N.evo maintain epithelial characteristics. d, Representative images of metaphase chromosome spreads of 2N.evo and 4N.evo and quantification of chromosome number by karyotyping (∼30 chromosome spreads were quantified in each condition). 4N.evo cells have a near-tetraploid karyotype. e, Centrosome amplification in diploid cells (2N or 2N.evo) newly generated tetraploid cells (4N) and evolved tetraploid cells (4N.evo). f, Quantification of the percentage of aneuploid cells in the ‘evolved’ cells. The 4N.evo cells are aneuploidy despite their near-tetraploid genomes (∼30 chromosome spreads were quantified in each condition). g, Quantification of centrosome amplification of 4N.evo cells overexpressing PLK4. Error bars represent mean ± s.e. from 3 independent experiments. h, Quantification of the invasive acini in 4N.evo cells after PLK4 overexpression. This experiment serves as a control to demonstrate that the 4N.evo cells retain their ability to amplify centrosomes and, after centrosome amplification, retain the capacity to form invasive acini. Error bars represent mean ± s.e. from 3 independent experiments. P value derived from unpaired two-tailed Student’s t-test (**P < 0.005).

Extended Data Figure 3 Characterization of invasive structures in cells with extra centrosomes.

a, F-actin and microtubules in invasive protrusions: F-actin (red), microtubules (α-tubulin, green) and DNA (blue). Insets show higher magnification images of the invasive protrusions. Scale bar, 10 μm. b, Fibronectin at invasive protrusions: cells were stained for F-actin (red), fibronectin (green) and DNA (blue). Scale bar, 10 μm. c, Fraction of invasive acini in 3D cultures after treatment with the broad spectrum matrix metalloprotease (MMP) inhibitor, marimastat (BB-2516). Error bars represent mean ± s.e. from 3 independent experiments. P value derived from unpaired two-tailed Student’s t-test (**P < 0.005). d, Images from videos of PLK4 overexpression cells (Supplementary Video 3), showing nuclei (labelled with H2B–GFP) migrating into an invasive protrusion (red arrows). Time scale shown as h:min. Scale bar, 20 μm. e, Multiple cells can migrate into invasive protrusions. Cells were stained for F-actin (red), laminin-V (green) and DNA (blue). Red arrows mark cells that migrated into the invasive protrusion. Scale bar, 10 μm. f, Western blot showing levels of E-cadherin, N-cadherin and vimentin in cells with (+Dox) and without (−Dox) extra centrosomes before and after 4 days in 3D culture. The western blots show that, unlike cells treated with TGF-β, cells with extra centrosomes do not acquire a canonical epithelial–mesenchymal transition (EMT) phenotype. We do note a small increase in the levels of vimentin in cells with extra centrosomes before plating in 3D cultures. Dox treatment was given for 48 h before 3D cultures in all experiments.

Extended Data Figure 4 Similarity between cells with centrosome amplification and cells with oncogene-induced invasion.

Cells were stained for F-actin (red), laminin-V (green) and DNA (blue). Similarity between the invasive protrusions of cells with extra centrosomes and the ones generated by cells overexpressing ERBB2, as previously reported4,44. In both conditions, invasive protrusions are characterized by the formation of actin-rich protrusions that are accompanied by degradation of the basement membrane. Scale bar, 10 μm.

Extended Data Figure 5 Reversine treatment induces aneuploidy but not invasive acini in MCF10A cells.

a, Quantification of the chromosome number in cells after treatment with with 0.1 μM of reversine (Rev) for 24 h before and after 4 days in 3D cultures (∼20 chromosome spreads were quantified in each condition). The concentration of reversine used does not induce cytokinesis failure and therefore would not induce centrosome amplification by inducing tetraploidy. b, Fraction of invasive acini after MCF10A cells are treated with reversine. Increased aneuploidy from reversine treatment does not induce invasion. Error bars represent mean ± s.e. from 3 independent experiments. c, Images of 3D cultures after treatment with reversine showing normal appearing acini. Scale bar, 50 μm. d, SNP analysis of MCF10A cells (without centrosome amplification) compared with Human Reference Genomic DNA 103 from Affymetrix. Previously reported genomic alterations in MCF10A cells can be detected in our analysis, namely: +5q, +6p and +8q.

Extended Data Figure 6 Invasive protrusions from 3D cultures of MCF10A cells with extra centrosomes are not an indirect consequence of altered cilia signalling, increased p53 expression or defects in centrosome polarization.

a, Cells in 2D were stained for pericentrin (green, inset), acetylated tubulin (red, inset) and DNA (blue). Cells were arrested for 48 h in G1 to induce primary cilium formation. Note that even in this case most of the cells do not form cilia. This is expected because MCF10A cells have limited proficiency for cilia formation, with only ∼7% of the cells assembling cilia even after 7 days of serum starvation45. Cells in 3D were stained for centrin (GFP, green inset), acetylated tubulin (red, inset) and DNA (blue). Cells do not form cilia after 4 days in 3D cultures. This is expected because, unlike MDCK cells, MCF10A cells do not have a discernable apical polarity and lumen after 4 days in 3D cultures and thus are unlikely to form primary cilium at this time. b, As expected17, centrosome amplification in MCF10A cells induces modest p53 activation. Note that this degree of p53 activation has a minor effect on the proliferation of MCF10A cells (Extended Data Figure 1d). Expression of PLK4 and PLK41–608 was induced by Dox for the indicated times: 0, 24, 48 and 72 h. c, Western blotting showing the levels of induction of p53 after doxorubicin treatment (200 ng ml−1 for 4 h) in control and p53-depleted cells, demonstrating that the p53 shRNA efficiently prevents p53 activation. d, Fraction of acini with invasive protrusions from cells with (+Dox) or without (−Dox) centrosome amplification after depletion of p53. The ability of cells to form invasive acini is not significantly affected by their p53 status. Error bars represent mean ± s.e. from 3 independent experiments. P values were derived from unpaired two-tailed Student’s t-test (***P < 0.0005; *P < 0.05). e, Cells were stained for α-catenin (green), γ-tubulin (red), DNA (blue) and the fibronectin micro-pattern visualized in red. Dashed boxes outline the centrosomes. Note that after PLK4 overexpression, extra centrosomes (clustered in interphase) are correctly positioned towards the cell–cell junction (similar to the control) even when the junction is defective, suggesting that centrosome amplification is not impairing the polarity axis of these cells. f, Centrosome positioning is not altered in cells with (+Dox) and without (control) extra centrosomes. Left image shows representative images of cells showing centrosomes (γ-tubulin) in relation to adherens junctions (α-catenin). Right image shows scheme with quantification of the fraction of centrosomes at the indicated positions on the micropatterns (see Fig. 3a). Note that the position of centrosomes in cells with centrosome amplification does not differ from that in control cells. Cells were plated on the patterns 48 h after induction of centrosome amplification with PLK4.

Extended Data Figure 7 Centrosome amplification induces cell scattering and Rac activation.

a, Quantification of number of cells with (+Dox) or without (−Dox) extra centrosomes that remain as pairs within 10 h time after mitosis. For –Dox n = 180; +Dox n = 98. Cell scattering occurred in most of the cases within the first 2 h after mitosis. Cells were imaged on 2D substratum. Similar results were obtained with fixed cells. Error bars represent mean ± s.e. from 3 independent experiments. b, Still images from videos showing examples of cells that stay together (−Dox) or move apart (+Dox). Time scale shown as h:min. c, Western blot showing levels of p120 catenin in cells with (+Dox) and without (−Dox) extra centrosomes in 2D and 3D cultures. d, Western blot from a pull-down experiment to detect GTP-bound Rac1 in HaCaTs cells. Graph shows quantification of active Rac1 from pull-down experiments. Error bars represent mean ± s.e. from 3 independent experiments. e, Western blot from a pull-down experiment to detect GTP-bound Rac1 in 16HBE cells. Graph shows quantification of active Rac1 from pull-down experiments. Error bars represent mean ± s.e. from 2 independent experiments. f, FRET control demonstrating increased CFP emission after photobleaching of the YFP fluorophore at an excitation wavelength of 510 nm for 10 min in MCF-10A single cells expressing Raichu-Rac. g, Levels of active Rac1 measured by FRET in cells overexpression PLK41–608. For –Dox, n = 25; +Dox, n = 22. Error bars represent mean ± s.e. h, Western blot from a pull-down experiment to detect GTP-bound RhoA in MCF10A cells showing decrease RhoA activity in cells with extra centrosomes. i, Levels of active Rac1 measured by FRET in cells with extra centrosomes treated with the Rac1 inhibitor NSC23766, demonstrating that NSC23766 inhibits Rac1 activation in cells with extra centrosomes. For −NSC23766, n = 37; +NSC23766, n = 36. Error bars represent mean ± s.e. All the P values were derived from unpaired two-tailed Student’s t-test (***P < 0.0005; **P < 0.005; *P < 0.05). Scale bar, 10 μm.

Extended Data Figure 8 Cell–cell adhesion defects caused by centrosome amplification can be observed in tetraploid cells and can be suppressed by Arp2/3 complex inhibition.

a, Western blot from a pull-down experiment to detect GTP-bound Rac1 in tetraploid MCF10A cells. b, Distribution of the cell–cell junction angles (left) and size (right) in the indicated tetraploid cells, with or without treatment with the Rac1 inhibitor, NSC23766. Note that tetraploid cells with extra centrosomes (4N) have a striking defect in junction positioning by comparison with tetraploid cells with normal centrosome number (4N.evo). This severe phenotype is only partially rescued by Rac1 inhibition. For 4N.evo, n = 106; 4N, n = 70; NCS23766, n = 47. Error bars represent mean ± s.e. c, Examples of cell doublets with (+Dox) or without (−Dox) centrosome amplification on the fibronectin micro-patterns. Cells were stained for F-actin (red), β-catenin (green), DNA (blue). d, Examples of cell doublets with (+Dox) or without (−Dox) centrosome amplification on the fibronectin micro-patterns treated with the Arp2/3 inhibitor (CK-666). Cells were stained for F-actin (red), β-catenin (green), DNA (blue). e, Distribution of the junction angle and quantification of the junction size in cells with extra centrosomes treated with 50 μM of Arp2/3 inhibitor (CK-666) for 6 h. Cells were analysed 48 h after Dox treatment. For –Dox, n = 251; +Dox, n = 160; CK666, n = 168. Error bars represent mean ± s.e. All the P values were derived from unpaired Student’s two-tailed t-test (***P < 0.0005; **P = 0.005). Scale bar, 10 μm.

Extended Data Figure 9 Quantification of centrosomal γ-tubulin in cells with extra centrosomes and levels of active Rac1 in the absence of EGF.

a, Images of centrosomes from interphase MCF10A cells stained for γ-tubulin. Boxes represent the region for measurement of centrosomal γ-tubulin signal (inside) and background (area between inner and outer boxes). b, Method used to determine the integrated fluorescent intensity of centrosomal γ-tubulin, as previously described41. c, Measurement of γ-tubulin intensity at the centrosomes showing that in interphase, increased centriole number is sufficient to increase γ-tubulin levels at the centrosomes whereas increased ploidy per se does not (4Nevo). For PLK4 –Dox, n = 60; PLK4 +Dox, n = 49; 4N.evo. PLK4 –Dox, n = 34; 4N.evo. PLK4 +Dox, n = 35. Error bars represent mean ± s.e. d, Increased Rac1 activity in cells with extra centrosomes can be detected in arrested cells deprived of EGF. Quantification of Rac1 activity by FRET in single cells with (+Dox) and without (−Dox) extra centrosomes in the absence of EGF, and examples of FRET images of cells in the absence of EGF. For PLK4 −Dox, n = 36; PLK4 +Dox, n = 47. Error bars represent mean ± s.e. Scale bar, 10 μm. e, FACS profiles of control (−Dox) and cells with extra centrosomes (+Dox) after 48 h of Dox treatment showing that there is not major difference in the cell cycle profiles of these cells. Note that at this time point centrosome amplification does not produce a striking defect in cell proliferation (Extended Data Fig. 1d). All the P values were derived from unpaired two-tailed Student’s t-test (**P < 0.005; ***P < 0.0005).

Extended Data Figure 10 Depletion of CEP192 suppresses the invasive properties of cells with centrosome amplification.

a, Scheme of the experimental design to induce centrosome amplification in cells depleted of CEP192 by siRNA. Transient overexpression of PLK4 is induced 6 h after siRNA to allow efficient centrosome overduplication. As expected, after depletion of CEP192 for 48 h, cells are partially compromised in their ability to overduplicate centrosomes after PLK4 overexpression46. b, Western blot showing efficient depletion of CEP192 after 48 h treatment of cells with CEP192 siRNA. c, Western blot showing partial depletion of CEP192 by shRNA. d, Quantification of centrosomal γ-tubulin after depletion of CEP192 by siRNA for 48 h. Similar results were observed with CEP192 esiRNA (not shown). It is notable that at least for a three-day period, cells remain viable after CEP192 knockdown. For control siRNA, n = 22; CEP192 siRNA, n = 20. Error bars represent mean ± s.e. Quantification of centrosome amplification after depletion of CEP192 by siRNA (e) or shRNA (f). Error bars represent mean ± s.e. from 3 independent experiments. g, Bright field images of acini after 4 days in 3D culture, demonstrating that partial CEP192 depletion by shRNA does not significantly impair cell growth or the formation of acini. Red arrows indicate the invasive acini. h, Quantification of PLK4-mediated centrosome amplification in cells depleted of CEP192 after 4 days in 3D cultures showing that these cells still carry extra centrosomes. Error bars represent mean ± s.e. from 3 independent experiments. Images show normal acini displaying centrosome amplification after partial knockdown of CEP192. Cells were stained for F-actin (red), centrioles (centrin1-GFP, green) and DNA (blue). Scale bar, 10 μm. i, Levels of active Rac1 measured by FRET after CEP192 depletion. For ctr.siRNA –Doc, n = 51; ctr.siRNA +Dox, n = 35; CEP.siRNA –Dox, n = 53; CEP.siRNA +Dox, n = 37. Error bars represent mean ± s.e. All the P values were derived from unpaired two-tailed Student’s t-test (*P < 0.05; **P < 0.005).

Supplementary information

Timelapse phase contrast imaging of acini lacking centrosome amplification (-Dox)

3-D culture was for 4 days prior to initiating imaging. Images were acquired over 20hrs with images acquired every 4 minutes. (MOV 1518 kb)

Timelapse imaging of an acinus with centrosome amplification as in Supplementary Movie 1

Note that invasive protrusions are dynamic. (MOV 1527 kb)

Multiple cells can migrate into an invasive protrusion from acini with multiple centrosomes

Imaging was as in Supplementary Movie S2 except that images were acquired for 8hrs and nuclei were visualized by fluorescence imaging every 4 minutes to detect nuclei marked by H2B-GFP. Phase contrast images were acquired at each timepoint with the fluorescence images. (MOV 234 kb)

Time lapse imaging of dividing single cells in 2-D substrates that stay together after mitosis

Images were acquired for 15hrs and nuclei were visualized by fluorescence imaging every 4 minutes to detect nuclei marked by H2B-GFP. Phase contrast images were acquired at each timepoint with the fluorescence images. Note that this time lapse represents only 5rs of the original 15hrs movie. (MOV 222 kb)

Time lapse imaging of dividing single cells on 2-D substrates that move apart after mitosis

Images were acquired for 15hrs and nuclei were visualized by fluorescence imaging every 4 minutes to detect nuclei marked by H2B-GFP. Phase contrast images were acquired at each timepoint with the fluorescence images. Note that this time lapse represents only 5rs of the original 15hrs movie. (MOV 196 kb)

Time lapse imaging of a monolayer of MCF10A cells expressing Raichu-Rac to visualize cell-cell boundaries

Images were acquired every 5 minutes for 12.4 hrs to monitor cell-cell edges interactions marked by Raichu-Rac-CFP. Scale bar :10µm (MOV 657 kb)

Time lapse imaging of a monolayer of MCF10A cells with centrosome amplification (+Dox) expressing Raichu-Rac-CFP as in Supplementary Movie 6

Overlapping regions between adjacent cells are marked by the white arrows. Scale bar :10µm (MOV 581 kb)

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Godinho, S., Picone, R., Burute, M. et al. Oncogene-like induction of cellular invasion from centrosome amplification. Nature 510, 167–171 (2014). https://doi.org/10.1038/nature13277

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