Hsp72 and Nek6 Cooperate to Cluster Amplified Centrosomes in Cancer Cells
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Published OnlineFirst July 18, 2017; DOI: 10.1158/0008-5472.CAN-16-3233 Cancer Molecular and Cellular Pathobiology Research Hsp72 and Nek6 Cooperate to Cluster Amplified Centrosomes in Cancer Cells Josephina Sampson1, Laura O'Regan1, Martin J.S. Dyer2, Richard Bayliss3, and Andrew M. Fry1 Abstract Cancer cells frequently possess extra amplified centrosomes Unlike some centrosome declustering agents, blocking Hsp72 clustered into two poles whose pseudo-bipolar spindles exhib- or Nek6 function did not induce formation of acentrosomal it reduced fidelity of chromosome segregation and promote poles, meaning that multipolar spindles were observable only genetic instability. Inhibition of centrosome clustering triggers in cells with amplified centrosomes. Inhibition of Hsp72 in multipolar spindle formation and mitotic catastrophe, offer- acute lymphoblastic leukemia cells resulted in increased mul- ing an attractive therapeutic approach to selectively kill cells tipolar spindle frequency that correlated with centrosome with amplified centrosomes. However, mechanisms of centro- amplification, while loss of Hsp72 or Nek6 function in non- some clustering remain poorly understood. Here, we identify a cancer-derived cells disturbs neither spindle formation nor new pathway that acts through NIMA-related kinase 6 (Nek6) mitotic progression. Hence, the Nek6–Hsp72 module repre- and Hsp72 to promote centrosome clustering. Nek6, as well as sents a novel actionable pathway for selective targeting of its upstream activators polo-like kinase 1 and Aurora-A, tar- cancer cells with amplified centrosomes. Cancer Res; 77(18); geted Hsp72 to the poles of cells with amplified centrosomes. 4785–96. Ó2017 AACR. Introduction are nucleated (11, 12). The g-TuRC is concentrated at centrosomes through binding to components of the pericentriolar material The mitotic spindle is bipolar in nature as it is organized around (PCM), a highly ordered matrix of coiled-coil proteins that two poles, each possessing a single centrosome (1, 2). However, surrounds and is scaffolded by a pair of centrioles (13). The the majority of cancer cells have extra or "amplified" centrosomes centrioles are composed of nine triplets of highly stable micro- that would be expected to form multipolar spindles, leading to tubules organized into a polarized barrel that is duplicated mitotic catastrophe and cell death (3, 4). Cancer cells solve this once per cell cycle. Hence, cells in G have two centrioles, while problem by either inactivating the extra centrosomes or clustering 1 cells in late G have four centrioles (14). These centrioles them into two poles to form a pseudo-bipolar spindle (5). The 2 remain in close proximity during interphase due to proteina- added time taken to organize such a spindle and presence of more ceous ties that link them together(15).Althougheach centriole than two centrosomes in early mitosis favors generation of is associated with its own PCM, the close juxtaposition of new merotelic microtubule–chromosome attachments that are poorly centrioles with their parental centrioles in S–G mean that only recognized by the spindle assembly checkpoint (SAC; refs. 6, 7). 2 two distinct PCM clouds are visible both before and after Hence, amplified centrosomes are not only tolerated but promote duplication. Hence, when scoring centrosome amplification, chromosome segregation errors, genome instability, and cancer we use the criteria that cells should have more than two PCM progression (8–10). foci and more than four centrioles. Centrosomes act as the dominant microtubule organizing Experimental studies have revealed a number of microtu- centers (MTOC) in proliferating animal cells because they recruit bule-based processes that promote centrosome clustering, the g-tubulin ring complex (g-TuRC) from which microtubules including motor protein activity, microtubule dynamics, and microtubule attachments to the centrosome, chromosomes, and cell cortex (16–18). In addition, the extra time taken to 1Department of Molecular and Cell Biology, University of Leicester, Leicester, organize a pseudo-bipolar spindle in cells with amplified United Kingdom. 2Ernest and Helen Scott Haematological Research Institute, centrosomes means that clustering requires cells to have a 3 University of Leicester, Leicester, United Kingdom. Astbury Centre for Struc- robust SAC that can delay anaphase onset until complete tural Molecular Biology, Faculty of Biological Sciences, University of Leeds, chromosome congression has been achieved (16, 19). Targeting Leeds, United Kingdom. these different clustering processes could provide a selective Note: Supplementary data for this article are available at Cancer Research approach to killing cancer cells with amplified centrosomes Online (http://cancerres.aacrjournals.org/). (20). However, the underlying mechanisms remain far from Corresponding Author: Andrew M. Fry, University of Leicester, Lancaster Road, understood. Leicester LE1 9HN, United Kingdom. Phone: 44-0116-229-7069; Fax: 44-116-229- Uncoupling of the centrosome duplication cycle from the cell 7018; E-mail: [email protected] cycle can result from altered activity of tumor suppressor genes doi: 10.1158/0008-5472.CAN-16-3233 and oncogenes and is a major route to centrosome amplification Ó2017 American Association for Cancer Research. in cancer cells (21–23). This could arise from changes in gene www.aacrjournals.org 4785 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst July 18, 2017; DOI: 10.1158/0008-5472.CAN-16-3233 Sampson et al. expression of centrosome duplication regulators caused by (Hsp70i, 10 mmol/L; Tocris Bioscience); Griseofulvin (10 deregulated signal transduction pathways. Alternatively, it could mmol/L; Sigma); MLN8054 (Aurora-Ai; 500 nmol/L; MedChem be due to reduced integrity of checkpoints, such as the SAC or Express); BI-2536 (Plk1i; 100 nmol/L; MedChem Express). DNA damage checkpoints, which disturb the timing of cell-cycle Control cells were treated with the same volume of DMSO. progression and perturb coordination with the centrosome dupli- For microtubule regrowth, cells were incubated for 16 hours cation cycle. Cancer cells also need to tolerate the presence of with 500 ng/mL nocodazole (Sigma) followed by treatment amplified centrosomes and destabilization of p53 through inac- with the 10 mmol/L VER-155008 for 2 hours. M-phase arrested tivation of the Hippo pathway may be a common mechanism in cells were prepared by mitotic shake-off after 16 hours of this regard (24). nocodazole treatment. Oncogenesis is associated with proteotoxic stress that leads to induction of heat shock proteins (HSP) required to maintain Fixed and live-cell microscopy protein folding and homeostasis (25, 26). As such HSPs represent Adherent cells were grown on acid-etched glass coverslips while cancer targets that are not necessarily restricted to tumors with suspension cells were seeded onto Superfrost Plus glass slides particular driver mutations (27, 28). Using RNAi depletion and (Thermo). Cells were fixed with ice-cold methanol before being chemical inhibitors, we recently found that Hsp72 is essential for processed for immunofluorescence microscopy as previously mitotic spindle assembly in cancer cells, promoting stabilization described (29, 32, 33). For Hsp72 staining, cells were incubated of kinetochore-associated microtubules, (K)-fibers, through for 30 seconds in pre-extraction buffer (60 mmol/L Pipes, 25 recruitment of the chTOG–TACC3 complex (29). This function mmol/L Hepes, pH7.4, 10 mmol/L EGTA, 2 mmol/L MgCl , and of Hsp72 is dependent upon its phosphorylation by the Nek6 2 1% Triton X-100) prior to fixation. Primary antibodies were mitotic kinase. As Hsp72 and Nek6 are required for K-fiber against a-tubulin (0.3 mg/mL; Sigma), g-tubulin (0.5 mg/mL; stability and robust mitotic spindle assembly, we reasoned that Sigma), centrin-2 (N-17; 0.4 mg/mL; Santa Cruz Biotechnology), they might also be required for clustering of amplified centro- CEP135 (2 mg/mL; OriGene), pericentrin (2 mg/mL; Abcam), somes. Here, we set out to test this hypothesis using fixed and CenpA (2 mg/mL; Abcam), Hsp72 (0.8 mg/mL; C92F3A-5; Enzo time-lapse imaging of MDA-MB-231 (human breast carcinoma) Life Sciences), BubR1 (10 mg/mL; Abcam) and phospho-Histone and NIE-115 (mouse neuroblastoma) cell lines that harbor H3 (2 mg/mL; Abcam). DNA was stained with 0.8 mg/mL Hoechst amplified centrosomes and exhibit centrosome clustering activity 33258. Secondary antibodies were Alexa Fluor-488 and -594 goat (16, 30, 31). Our data reveal a new pathway of centrosome anti-rabbit and goat anti-mouse IgGs (1 mg/mL; Invitrogen). clustering and lead us to propose a therapeutic role for Hsp72 Imaging was performed on a Leica TCS SP5 confocal microscope and/or Nek6 inhibitors as selective declustering agents in cancer equipped with a Leica DMI 6000B inverted microscope using a cells with amplified centrosomes. 63x oil objective (numerical aperture, 1.4). Z-stacks comprising of 30–50 Â 0.3-mm sections were acquired. Time-lapse imaging was Materials and Methods performed on a Leica TCS SP5 confocal microscope equipped with a Leica DMI 6000B inverted microscope using a 63Â oil Cell culture, synchronization, transfection, and drug objective (numerical aperture, 1.4). Cells were cultured in glass- treatments bottomed culture dishes (MatTek Corp.) and maintained at 37C All media were from Invitrogen and supplemented with 10% in an atmosphere supplemented with CO using a microscope