ORIGINAL ARTICLE N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore–microtubule dynamics M Orticello 1,7 , M Fiore 1,7 , P Totta 1,8 , M Desideri 2 , M Barisic 3 , D Passeri 4 , J Lenzi 5 , A Rosa 5 , A Orlandi 4 , H Maiato 3,6 , D Del Bufalo 2 and F Degrassi 1 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. Oncogene (2015) 34, 3325–3335; doi:10.1038/onc.2014.265; published online 18 August 2014 INTRODUCTION Highly Expressed in Cancer protein 1 (Hec1) 1 is a constituent of the evolutionary conserved Ndc80 complex, the primary site of interaction between kinetochores (KTs) and microtubules (MTs). 2 The Ndc80 complex localizes at KTs and is composed of four subunits: Hec1, Nuf2, Spc24, and Spc25. 2 This complex is required for accurate chromosome segregation in mitosis, as it is essential for generating bipolar end-on KT–MT attachments, which are responsible for the faithful anaphase segregation of sister chromatids. 2,3 Because of the stochastic nature of KT–MT encounters, during the early stages of mitosis erroneous KT–MT interactions frequently occur, which are subsequently modified by a process of error correction. This process is governed by the mitotic kinase aurora B that phosphorylates the Hec1 N-terminal tail 4 and several other substrates that modulate KT–MT attach- ment stability. 5–8 HEC1 mRNA is overexpressed in 60 out of the 65 data sets of different human cancer tissues in the Oncomine database (www. oncomine.org), and elevated Hec1 expression is associated with negative prognosis in multiple cancer types. 9–11 Moreover, HEC1 mRNA and protein are found overexpressed in lung and colorectal cancer cells. 10,12 The crucial role of the Ndc80 complex in chromosome segregation during mitosis, the recurrent HEC1 upregulation in different human cancers and the dependence of Hec1 upregulation on pRb deficiency 13 suggest that HEC1 deregulation may be an important step in the multistage process of tumorigenesis. Indeed, Hec1 overexpression in an inducible mouse model has been shown to promote chromosome instability in embryonic fibroblasts and tumour formation in different mouse tissues. 14 Compelling evidence has highlighted a double role of chromo- some instability in cancer, leading to a model in which low chromosome instability results in a slight growth advantage and/ or tumour promotion and thereby promotes cancer cell transfor- mation, whereas high chromosome instability leads to cell death and acts as a tumour-suppressor mechanism. 15,16 Consequently, the idea of promoting cell death by inducing massive aneuploidy at mitotic division has been proposed as a therapeutic strategy to selectively eliminate highly proliferating tumour cells. 17,18 We previously reported that expression of Hec1 fused at its N-terminus—an MT interacting domain—with the enhanced green fluorescent protein (EGFP) tag (EGFP-Hec1) acts as a 1 Institute of Biology, Molecular Medicine and Nanobiotechnology, CNR National Research Council, Rome, Italy; 2 Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy; 3 Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; 4 Anatomic Pathology Institute, Tor Vergata University, Rome, Italy; 5 Department of Biology and Biotechnology ‘Charles Darwin’, Sapienza University, Rome, Italy and 6 Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, Porto, Portugal. Correspondence: Dr F Degrassi, Institute of Biology, Molecular Medicine and Nanobiotechnology, CNR National Research Council, Via degli Apuli 4, Rome 00185, Italy. E-mail: francesca.degrassi@uniroma1.it 7 These authors contributed equally to this work. 8 Current address: Department of Science, Biomedical Science and Technology Section, ‘Roma Tre’ University, Rome, Italy. Received 6 December 2013; revised 11 June 2014; accepted 3 July 2014; published online 18 August 2014 Oncogene (2015) 34, 3325 – 3335 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc