Original Articles Polo-like kinase 1 inhibitor BI2536 causes mitotic catastrophe following activation of the spindle assembly checkpoint in non-small cell lung cancer cells Minji Choi a,b , Wootae Kim a,b , Min Gyeong Cheon a,b , Chang-Woo Lee c , Ja-Eun Kim a,b, * a Department of Pharmacology, School of Medicine, Kyung Hee University, Seoul 130-701, Korea b Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 130-701, Korea c Department of Molecular Cell Biology, Samsung Biomedical Research Institute, School of Medicine, Sungkyunkwan University, Suwon, Gyeonggi 440-746, Korea ARTICLE INFO Article history: Received 14 October 2014 Received in revised form 5 December 2014 Accepted 9 December 2014 Keywords: PLK1 BI2536 Non-small cell lung cancer Mitosis Spindle assembly checkpoint Mitotic catastrophe A B ST R AC T Polo-like kinase 1 (PLK1), a critical kinase that regulates multiple steps in mitosis, is overexpressed in diverse human cancers; thus many PLK1 inhibitors have been developed as potential cancer therapeu- tic agents. One of these compounds, the PLK1-specific inhibitor BI2536, has been investigated as a cytotoxic drug in several cancers, including lung cancer; however, the detailed mechanism by which BI2536 induces defects in cell proliferation of non-small cell lung cancer (NSCLC) has not yet been determined. We found that BI2536 treatment resulted in mitotic arrest due to improper formation of the mitotic spindle and mitotic centrosomes. The unattached kinetochores in BI2536-treated NSCLC cells activated the spindle assembly checkpoint (SAC). The prolonged activation of the SAC led to a type of apoptotic cell death re- ferred to as mitotic catastrophe. Finally, BI2536-treated NSCLC cells show a defect in cell proliferation. Overall, these data indicate that PLK1 inhibition via mitotic disruption represents a potential approach for the treatment of NSCLC. © 2014 Published by Elsevier Ireland Ltd. Introduction Mitosis is a critical cell cycle phase that passes one of each pair of sister chromatids to each daughter cell; thus precise orchestra- tion of mitosis is necessary for the maintenance of chromosomal stability in cells [1]. Aberrant mitotic progression leads to chromo- somal mis-segregation and can contribute to cancer development [2–4]. To prevent the steps of mitosis from occurring out of order, cells are equipped with safeguard systems, as follows: the antephase checkpoint, which controls entry into mitosis; the spindle assem- bly checkpoint (SAC), which senses kinetochores not attached to microtubules [5–7]; and the abscission checkpoint, which pre- vents the completion of cytokinesis in the presence of lagging chromosomes [8]. Among these, the SAC has been most exten- sively investigated. The SAC, also called the mitotic checkpoint, is required for proper chromosome segregation [5–7]. When one or more kinetochores are not attached to microtubules, the SAC is activated to delay the tran- sition from metaphase to anaphase until the cell can correct the aberrant kinetochore–microtubule attachment. In humans, the SAC is dynamically regulated by several key players including monopolar spindle protein 1 (Mps1), budding uninhibited by benzimidazoles 1 (Bub1), Bub3, Bub1-related 1 (BubR1), mitotic arrest-deficient protein 1 (Mad1), Mad2, Aurora B, and Polo-like kinase 1 (PLK1). SAC activation is initiated by the formation of the mitotic check- point complex (MCC) composed of Mad2, BubR1, Bub3, and Cdc20. Enrichment of MCC at unattached kinetochores inactivates Cdc20, a co-factor of the anaphase-promoting complex/cyclosome (APC/ C) E3 ubiquitin ligase, thereby blocking degradation of cyclin B and securin and delaying the onset of anaphase. Recruitment of SAC pro- teins and amplification of SAC signaling is also regulated by the Mps1, Bub1, BubR1, Aurora B, and PLK1 kinases [9,10]; thus SAC and its regulatory kinases act as a crucial cell cycle surveillance mecha- nism that protects the cell against chromosomal instability. The serine/threonine kinase PLK1 governs several mitotic steps, including entry into mitosis, centrosome maturation, bipolar spindle assembly, activation of APC/C by phosphorylation of early mitotic inhibitor 1 (Emi1), chromosome segregation, and mitotic exit [11–13]. PLK1 expression starts to rise in G2 and peaks at mitosis [14,15]. PLK1 is localized at centrosomes in G2/prophase, centrosomes and kinetochores in prometaphase, spindle poles and both aligned and unaligned kinetochores in metaphase, spindle microtubules in ana- phase, the central spindle in telophase, and the cleavage furrow and midbody during cytokinesis [14,16,17], suggesting that it plays roles Abbreviations: PLK1, Polo-like kinase 1; NSCLC, non-small cell lung cancer; SAC, spindle assembly checkpoint; MCC, mitotic checkpoint complex. * Corresponding author. Tel.: +82 2 961 0235; fax: +82 2 968 0560. E-mail address: jekim@khu.ac.kr (J-E. Kim). http://dx.doi.org/10.1016/j.canlet.2014.12.023 0304-3835/© 2014 Published by Elsevier Ireland Ltd. Cancer Letters ■■ (2014) ■■–■■ ARTICLE IN PRESS Please cite this article in press as: Minji Choi, Wootae Kim, Min Gyeong Cheon, Chang-Woo Lee, Ja-Eun Kim, Polo-like kinase 1 inhibitor BI2536 causes mitotic catastrophe fol- lowing activation of the spindle assembly checkpoint in non-small cell lung cancer cells, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.12.023 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Q1 Q2 Q3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90