Skip to main content

Advertisement

SpringerLink
Log in

N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore–microtubule dynamics

  • Original Article
  • Published:
Oncogene Submit manuscript

Abstract

Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT–microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT–MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT–MT attachment dynamics constitutes a powerful approach in cancer therapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Chen Y, Riley DJ, Chen P-L, Lee WH . HEC, a novel nuclear protein rich in leucine heptad repeats specifically involved in mitosis. Mol Cell Biol 1997; 17: 6049–6056.

    Article  CAS  Google Scholar 

  2. Ciferri C, Pasqualato S, Screpanti E, Varetti G, Santaguida S, Dos Reis G et al. Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex. Cell 2008; 133: 427–439.

    Article  CAS  Google Scholar 

  3. DeLuca JG, Musacchio A . Structural organization of the kinetochore-microtubule interface. Curr Opin Cell Biol 2012; 24: 48–56.

    Article  CAS  Google Scholar 

  4. DeLuca J, Gall WE, Ciferri C, Cimini D, Musacchio A, Salmon ED . Kinetochore microtubule dynamics and attachment stability are regulated by Hec 1. Cell 2006; 127: 969–982.

    Article  CAS  Google Scholar 

  5. Lan W, Zhang X, Kline-Smith SL, Rosasco SE, Barrett-Wilt GA, Shabanowitz J et al. Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity. Curr Biol 2004; 14: 273–286.

    Article  CAS  Google Scholar 

  6. Welburn JP, Vleugel M, Liu D, Yates JR 3rd, Lampson MA, Fukagawa T et al. Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface. Mol Cell 2010; 38: 383–392.

    Article  CAS  Google Scholar 

  7. Bakhoum SF, Thompson SL, Manning AL, Compton DA . Genome stability is ensured by temporal control of kinetochore-microtubule dynamics. Nat Cell Biol 2009; 1: 27–35.

    Article  Google Scholar 

  8. Manning AL, Bakhoum SF, Maffini S, Correia-Melo C, Maiato H, Compton DA . CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore-microtubule dynamics to promote mitotic progression and fidelity. EMBO J 2010; 29: 3531–3543.

    Article  CAS  Google Scholar 

  9. Glinsky GV, Berezovska O, Glinskii AB . Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest 2005; 115: 1503–1521.

    Article  CAS  Google Scholar 

  10. Hayama S, Daigo Y, Kato T, Ishikawa N, Yamabuki T, Miyamoto M et al. Activation of CDCA1-KNTC2, members of centromere protein complex, involved in pulmonary carcinogenesis. Cancer Res 2009; 66: 10339–10348.

    Article  Google Scholar 

  11. Bièche I, Vacher S, Lallemand F, Tozlu-Kara S, Bennani H, Beuzelin M et al. Expression analysis of mitotic spindle checkpoint genes in breast carcinoma: role of NDC80/HEC1 in early breast tumourigenicity, and a two-gene signature for aneuploidy. Mol Cancer 2011; 10: 23.

    Article  Google Scholar 

  12. Kaneko N, Miura K, Gu Z, Karasawa H, Ohnuma S, Sasaki H et al. siRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis. Biochem Biophys Res Commun 2009; 390: 1235–1240.

    Article  CAS  Google Scholar 

  13. Ferretti C, Totta P, Fiore M, Mattiuzzo M, Schillaci T, Ricordy R et al. Expression of the kinetochore protein Hec1 during the cell cycle in normal and cancer cells and its regulation by the pRb pathway. Cell Cycle 2010; 9: 4174–4182.

    Article  CAS  Google Scholar 

  14. Diaz-Rodríguez E, Sotillo R, Schvartzman JM, Benezra R . Hec1 overexpression hyperactivates the mitotic checkpoint and induces tumor formation in vivo. Proc Natl Acad Sci USA 2008; 105: 16719–16724.

    Article  Google Scholar 

  15. Janssen A, Medema RH . Genetic instability: tipping the balance. Oncogene 2013; 32: 4459–4470.

    Article  CAS  Google Scholar 

  16. Duijf PH, Benezra R . The cancer biology of whole-chromosome instability. Oncogene 2013; 32: 4727–4736.

    Article  CAS  Google Scholar 

  17. Janssen A, Kops GJ, Medema RH . Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells. Proc Natl Acad Sci USA 2009; 106: 19108–19113.

    Article  CAS  Google Scholar 

  18. Galimberti F, Thompson SL, Liu X, Li H, Memoli V, Green SR, DiRenzo J et al. Targeting the cyclin E-Cdk-2 complex represses lung cancer growth by triggering anaphase catastrophe. Clin Cancer Res 2010; 16: 109–120.

    Article  CAS  Google Scholar 

  19. Mattiuzzo M, Vargiu G, Totta P, Fiore M, Ciferri C, Musacchio A et al. Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1. PLoS ONE 2011; 6: e16307.

    Article  CAS  Google Scholar 

  20. Guimaraes GJ, Dong Y, McEwen BF, Deluca JG . Kinetochore-microtubule attachment relies on the disordered N-terminal tail domain of Hec1. Curr Biol 2008; 18: 1778–1784.

    Article  CAS  Google Scholar 

  21. Miller SA, Johnson ML, Stukenberg PT . Kinetochore attachments require an interaction between unstructured tails on microtubules and Ndc80 (Hec1). Curr Biol 2008; 18: 1785–1791.

    Article  CAS  Google Scholar 

  22. Martin-Lluesma S, Stucke VM, Nigg EA . Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 2002; 297: 2267–2270.

    Article  CAS  Google Scholar 

  23. DeLuca J, Howell BJ, Canman JC, Hickey JM, Fang G, Salmon ED . Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores. Curr Biol 2003; 13: 2103–2109.

    Article  CAS  Google Scholar 

  24. Gurzov EN, Izquierdo M . RNA interference against Hec1 inhibits tumor growth in vivo. Gene Therapy 2006; 13: 1–7.

    Article  CAS  Google Scholar 

  25. Li L, Yang L, Scudiero DA, Miller SA, Yu ZX, Stukenberg PT et al. Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells. Gene Therapy 2007; 14: 814–827.

    Article  CAS  Google Scholar 

  26. Daum JR, Potapova TA, Sivakumar S, Daniel JJ, Flynn JN, Rankin S et al. Cohesion fatigue induces chromatid separation in cells delayed at metaphase. Curr Biol 2011; 21: 1018–1024.

    Article  CAS  Google Scholar 

  27. Stevens D, Gassmann R, Oegema K, Desai A . Uncoordinated loss of chromatid cohesion is a common outcome of extended metaphase arrest. PLoS ONE 2011; 6: e22969.

    Article  CAS  Google Scholar 

  28. Vitale I, Galluzzi L, Castedo M, Kroemer G . Mitotic catastrophe: a mechanism for avoiding genomic instability. Nat Rev Mol Cell Biol 2011; 12: 385–392.

    Article  CAS  Google Scholar 

  29. Zhai Y, Kronebusch PJ, Borisy GG . Kinetochore microtubule dynamics and the metaphase-anaphase transition. J Cell Biol 1995; 131: 721–734.

    Article  CAS  Google Scholar 

  30. McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL . A bright and photostable photoconvertible fluorescent protein. Nat Methods 2009; 6: 131–133.

    Article  CAS  Google Scholar 

  31. Rosa A, Brivanlou AH . A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation. EMBO J 2011; 30: 237–248.

    Article  CAS  Google Scholar 

  32. Morlando M, Dini Modigliani S, Torrelli G, Rosa A, Di Carlo V et al. FUS stimulates microRNA biogenesis by facilitating co-transcriptional Drosha recruitment. EMBO J 2012; 31: 4502–4510.

    Article  CAS  Google Scholar 

  33. Lacoste A, Berenshteyn F, Brivanlou AH . An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells. Cell Stem Cell 2009; 5: 332–342.

    Article  CAS  Google Scholar 

  34. Sethi G, Pathak HB, Zhang H, Zhou Y, Einarson MB, Vathipadiekal V et al. An RNA interference lethality screen of the human druggable genome to identify molecular vulnerabilities in epithelial ovarian cancer. PLoS ONE 2012; 7: e47086.

    Article  CAS  Google Scholar 

  35. Brito DA, Rieder CL . Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr Biol 2006; 16: 1194–1200.

    Article  CAS  Google Scholar 

  36. Gascoigne KE, Taylor SS . Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs. Cancer Cell 2008; 14: 111–122.

    Article  CAS  Google Scholar 

  37. Huang HC, Shi J, Orth JD, Mitchison TJ . Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly. Cancer Cell 2009; 16: 347–358.

    Article  CAS  Google Scholar 

  38. Kwon M, Godinho SA, Chandhok NS, Ganem NJ, Azioune A, Thery M et al. Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev 2008; 22: 2189–2203.

    Article  CAS  Google Scholar 

  39. Maiato H, Logarinho E . Mitotic spindle multipolarity without centrosome amplification. Nat Cell Biol 2014; 16: 386–393.

    Article  CAS  Google Scholar 

  40. O’Connor MJ, Martin NMB, Smith GCM . Targeted cancer therapies based on the inhibition of DNA strand break repair. Oncogene 2007; 26: 7816–7824.

    Article  Google Scholar 

  41. Degenhardt Y, Lampkin T . Targeting Polo-like kinase in cancer therapy. Clin Cancer Res 2010; 16: 384–389.

    Article  CAS  Google Scholar 

  42. Frecha C, Szécsi J, Cosset FL, Verhoeyen E . Strategies for targeting lentiviral vectors. Curr Gene Ther 2008; 8: 449–460.

    Article  CAS  Google Scholar 

  43. Zhou Q, Buchholz CJ . Cell type specific gene delivery by lentiviral vectors: new options in immunotherapy. Oncoimmunology 2013; 2: e22566.

    Article  Google Scholar 

  44. Schäfer J, Höbel S, Bakowsky U, Aigner A . Liposome-polyethylenimine complexes for enhanced DNA and siRNA delivery. Biomaterials 2010; 31: 6892–6900.

    Article  Google Scholar 

  45. Singh J, Michel D, Chitanda JM, Verrall RE, Badea I . Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles. J Nanobiotechnology 2012; 10: 7.

    Article  CAS  Google Scholar 

  46. Wang YQ, Su J, Wu F, Lu P, Yuan LF, Yuan WE et al. Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery. Int J Nanomedicine 2012; 7: 693–704.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010; 464: 1067–1070.

    Article  CAS  Google Scholar 

  48. Senzer N, Nemunaitis J, Nemunaitis D, Bedell C, Edelman G, Barve M et al. Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Mol Ther 2013; 21: 1096–1103.

    Article  CAS  Google Scholar 

  49. Manning AL, Compton DA . Mechanisms of spindle-pole organization are influenced by kinetochore activity in mammalian cells. Curr Biol 2007; 17: 260–265.

    Article  CAS  Google Scholar 

  50. Logarinho E, Maffini S, Barisic M, Marques A, Toso A, Meraldi P et al. CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment. Nat Cell Biol 2012; 14: 295–303.

    Article  CAS  Google Scholar 

  51. Kabeche L, Compton DA . Cyclin A regulates kinetochore microtubules to promote faithful chromosome segregation. Nature 2013; 502: 110–113.

    Article  CAS  Google Scholar 

  52. Cimini D, Wan X, Hirel CB, Salmon ED . Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr Biol 2006; 16: 1711–1718.

    Article  CAS  Google Scholar 

  53. Bakhoum SF, Genovese G, Compton DA . Deviant kinetochore microtubule dynamics underlie chromosomal instability. Curr Biol 2009; 19: 1937–1942.

    Article  CAS  Google Scholar 

  54. Zasadil LM, Andersen KA, Yeum D, Rocque GB, Wilke LG, Tevaarwerk AJ et al. Cytotoxicity of paclitaxel in breast cancer is due to chromosome missegregation on multipolar spindles. Sci Transl Med 2014; 6: 229ra43.

    Article  Google Scholar 

  55. Meraldi P, Draviam VM, Sorger PK . Timing and checkpoints in the regulation of mitotic progression. Dev Cell 2004; 7: 45–60.

    Article  CAS  Google Scholar 

  56. Wandke C, Barisic M, Sigl R, Rauch V, Wolf F, Amaro AC et al. Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis. J Cell Biol 2012; 198: 847–863.

    Article  CAS  Google Scholar 

  57. Orlandi A, Ferlosio A, Ciucci A, Francesconi A, Lifschitz-Mercer B, Gabbiani G et al. Cellular retinol binding protein-1 expression in endometrial hyperplasia and carcinoma: diagnostic and possible therapeutic implications. Mod Pathol 2006; 19: 797–803.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Live-cell imaging experiments were performed at the Nikon Reference Centre, CNR Institute of Biology, Molecular Medicine and Nanobiotechnology. We thank Giulia Guarguaglini and Patrizia Lavia for advice and support with live cell analysis. We also thank Giulia Vargiu for her contribution in the initial phases of the work, Enrico Cundari for helpful comments and suggestions, Antonio Pereira for expert advice and software update in the statistical analysis of MT dynamics data and Stephan Geley for mEOS-tubulin vector. This work was partially supported by a grant from the Italian Association for Cancer Research to DDB (IG 14100). Work in HM's laboratory is funded by FCOMP-01-0124-FEDER-015941 (PTDC/SAU-ONC/112917/2009) through COMPETE and Fundação para a Ciência e a Tecnologia of Portugal, the Human Frontier Science Program and PRECISE grant from the European Research Council.

Author information

Author notes

  1. P Totta

    Present address: 8Current address: Department of Science, Biomedical Science and Technology Section, ‘Roma Tre’ University, Rome, Italy.,

  2. M Orticello and M Fiore: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Biology, Molecular Medicine and Nanobiotechnology, CNR National Research Council, Rome, Italy

    M Orticello, M Fiore, P Totta & F Degrassi

  2. Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy

    M Desideri & D Del Bufalo

  3. Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

    M Barisic & H Maiato

  4. Anatomic Pathology Institute, Tor Vergata University, Rome, Italy

    D Passeri & A Orlandi

  5. Department of Biology and Biotechnology ‘Charles Darwin’, Sapienza University, Rome, Italy

    J Lenzi & A Rosa

  6. Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, Porto, Portugal

    H Maiato

Authors
  1. M Orticello
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Fiore
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P Totta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Desideri
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M Barisic
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D Passeri
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J Lenzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A Rosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. A Orlandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. H Maiato
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. D Del Bufalo
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. F Degrassi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to F Degrassi.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Orticello, M., Fiore, M., Totta, P. et al. N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore–microtubule dynamics. Oncogene 34, 3325–3335 (2015). https://doi.org/10.1038/onc.2014.265

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2014.265

  • Springer Nature Limited

This article is cited by

Search

Navigation