PNUTS) Regulates Aurora Kinases and Mitotic Progression Feifei Wang1,2,3, Ling Wang2, Laura A

Published OnlineFirst September 6, 2018; DOI: 10.1158/1541-7786.MCR-17-0670

Cell Cycle and Senescence Molecular Cancer Research Phosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates Aurora Kinases and Mitotic Progression Feifei Wang1,2,3, Ling Wang2, Laura A. Fisher2, Chunling Li3, Weidong Wang3, and Aimin Peng2

Abstract

Mitotic progression is regulated largely by reversible phos- associated proteins. PNUTS depletion suppresses the acti- phorylation events that are mediated by mitotic kinases vation of Aurora A/B kinases, and disrupts the spatiotem- and phosphatases. Protein phosphatase 1 (PP1) has been poral regulation of the chromosomal passenger complex shown to play a crucial role in regulation of mitotic entry, (CPC). PNUTS dynamically localizes to kinetochores, and progression, and exit. We previously observed, in Xenopus is required for the activation of the spindle assembly check- egg extracts, that phosphatase 1 nuclear targeting subunit point. Finally, PNUTS depletion sensitizes the tumor cell (PPP1R10/PNUTS) acts as a mitotic regulator by negatively response to Aurora inhibition, suggesting that PNUTS is a modulating PP1. This study investigates the role of PNUTS potential drug target in combination anticancer therapy. in mitotic progression in mammalian cells, and demon- strates that PNUTS expression is elevated in mitosis and Implications: Delineation of how PNUTS governs the depletion partially blocks mitotic entry. Cells that enter mitotic activation and function of Aurora kinases will mitosis after PNUTS knockdown exhibit frequent chromo- improve the understanding of the complex phospho-regu- some mis-segregation. Aurora A/B kinase complexes and lation in mitotic progression, and suggest new options to several kinetochore components are identiﬁed as PNUTS- enhance the therapeutic efﬁcacy of Aurora inhibitors.

Introduction therapy. Over the recent decade, numerous small-molecule inhibitors of Aurora kinases have been identified, and studies using Mitotic kinases, particularly cyclin-dependent kinase 1 these inhibitors demonstrated promising antitumor activities. (CDK1), polo-like kinase 1 (Plk1), and Aurora A/B, are well Many of these inhibitors have entered clinical trials for breast established as central regulators of mitosis. Activation of these cancer, prostate cancer, leukemia, and other malignancies (6, 8). kinases triggers the phosphorylation of various substrates to Protein phosphatases antagonize the action of kinases, but the modulate all aspects of mitotic cell reorganization and progres- function and regulation of mitotic phosphatases are considerably sion (1–4). Thus, regulated activation and deactivation of these less studied (1, 2, 4, 9–12). The two major groups of serine and kinases are defined as molecular events that dictate M-phase entry threonine phosphatases, protein phosphatase 1 (PP1) and 2A and exit. Among mitotic kinases, Aurora A and Aurora B kinases (PP2A), were both known to play important roles in mitotic phosphorylate a number of substrates to regulate the dynamics of regulation. For example, it has been shown that PP1 depho- centrosomes, microtubules, and chromatin during mitotic pro- sphorylates numerous mitotic factors and thereby regulates mitot- gression. Inactivation of Aurora A and B causes spindle defects, ic entry, metaphase–anaphase transition, chromatin condensa- chromosome missegregation, cytokinesis failure, and aneuploidy tion, and mitotic exit (13–15). A major gap in knowledge, (4–7). Owing to the crucial role of mitotic kinases in cell prolif- however, is how PP1 is regulated to achieve these dynamic and eration, and the profound toxicity of their inhibition, mitotic specific actions during mitotic progression. kinases are appreciated as potential drug targets for cancer Among the mitotic proteins dephosphorylated by PP1 were Aurora A and B kinases and their substrates, and the general role of PP1 as an antagonizer of Aurora kinases has been shown in 1Institute of Physical Science and Information Technology, Anhui University, multiple experimental systems (16–19). In yeast, the codeletion Hefei, China. 2Department of Oral Biology, College of Dentistry, University of of PP1/Glc7 with Aurora/Ipl1 rescued mitotic progression and cell Nebraska Medical Center, Lincoln, Nebraska. 3Institute of Hypertension, proliferation from Aurora/Ipl1 deletion (16). Similarly, PP1 Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. suppression in mammalian cells prevented mitotic defects Note: Supplementary data for this article are available at Molecular Cancer induced by the inhibition of Aurora B (17–19). The opposing Research Online (http://mcr.aacrjournals.org/). relationship between PP1 and Aurora is perhaps best illustrated in Corresponding Author: Aimin Peng, Department of Oral Biology, College of the regulation of spindle assembly checkpoint signaling at kine- Dentistry, University of Nebraska Medical Center, 40th & Holdrege, Lincoln, NE tochores. The spindle checkpoint is a surveillance mechanism that 68516. Phone: 402-472-5903; Fax: 402-472-2551; ensures the proper, bipolar kinetochore–microtubule attach- E-mail: [email protected] ments (20, 21). It has been shown that reversible phosphorylation doi: 10.1158/1541-7786.MCR-17-0670 of kinetochore components plays a pivotal role in governing 2018 American Association for Cancer Research. spindle attachment and the activation/deactivation of the spindle

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checkpoint (22). On one hand, centromeric Aurora B is respon- Immunoblotting and immunoprecipitation sible for the activation of the spindle checkpoint via both the SDS-PAGE and immunoblotting were performed as described recruitment of spindle checkpoint kinases, such as Mps1 and previously (36). For immunoprecipitation, anti-rabbit magnetic Bub1, and the subsequent phosphorylation of BubR1, Mad1, beads (New England Biolabs) were conjugated to the primary Ndc80/Hec1, Knl1, and other kinetochore proteins (21, 22). On antibody, and then incubated in cell lysates for 2 hours. The beads the other hand, several kinetochore components, especially Knl1, were collected using a magnet, washed, eluted with Laemmli yield PP1-binding activities, and were reported to recruit PP1 to sample buffer, and then analyzed by immunoblotting. kinetochores (19). After the proper spindle–kinetochore attachment, PP1 dephosphorylates Aurora B, Knl1, BubR1, and other Immunofluorescence and imaging kinetochore proteins to silence the spindle checkpoint and trigger Immunofluorescence was performed as described previously metaphase–anaphase transition (22). (37). Briefly, cells were fixed in a fixation buffer (3% formalde- Phosphatase 1 nuclear subunit (PNUTS), also known as hyde with 0.1% Triton X-100), washed, and blocked in a blocking PPP1R10, was originally described as a nuclear regulator of PP1 buffer (10% goat serum in PBS). The primary antibodies were that retains a portion of PP1 in the nucleus (23). PNUTS has been diluted in the blocking buffer, and incubated with the cells for implicated in transcription and RNA processing (24, 25), DNA 2 hours. The cells were then washed, and incubated with the Alexa – damage response and maintenance of telomere stability (26 30), Fluor 594 and 488 secondary antibodies (Invitrogen) for 1 hour. and modulation of RB and PTEN (31–33). In most cases, with the Imaging was performed using a Zeiss Axiovert 200M inverted fi exception of RNA Pol II, PNUTS acts to inhibit PP1 toward speci c fluorescence microscope at the UNMC Advanced Microscopy substrates. The earliest evidence that linked PNUTS to mitosis Core Facility. Live-cell imaging was performed using the Marianas in vitro came from the observation that PNUTS enhanced the Live Cell system based around a Zeiss Axiovert 200M microscope chromosome decondensation in a PP1-dependent manner (34). stand, and the SlideBook6 software (Intelligent Imaging Innova- Xenopus Interestingly, our recent study in egg extracts suggested a tions, Inc.). Images were collected with 10 objective lens mag- role of PNUTS as an essential regulator of mitotic progression. nification. Once the live-cell microscopy was completed, the Overexpression of PNUTS in Xenopus egg extracts inhibited mitot- captured images were loaded into SlideBook Reader Software ic exit. PNUTS depletion disrupted mitotic maintenance, whereas (Intelligent Imaging Innovations). codepletion of PP1 rescued the defect (35). The level of PNUTS oscillated in cycling extracts and peaked in mitosis. In this study, we sought to examine the role of PNUTS in human cells, and to Protein expression, pull-down, and mass spectrometry analysis reveal further mechanistic insights into how PNUTS governs MBP-tagged PNUTS was constructed and expressed as in our mitotic signaling. Our results confirmed a role of PNUTS in previous study (35). The recombinant protein was expressed in mammalian mitotic progression, characterized PNUTS-mediated BL21 bacterial cells and purified on amylose beads. For the pull- regulation of Aurora kinases and spindle checkpoint signaling, down assay, amylose beads conjugated with MBP–PNUTS were and suggested PNUTS as a potential target to enhance the cyto- incubated in Xenopus egg extracts that were prepared as in our toxicity of Aurora inhibitors. previous study (35). The beads were reisolated, washed, eluted, and then resolved by SDS-PAGE for immunoblotting or mass spectrometry (Taplin mass spectrometry facility, Harvard Medical Materials and Methods School, Boston, MA). Antibodies and other reagents Cdc27 antibody was purchased from BD Transduction Labo- ratories; Aurora A, Aurora B, Bub1, Cenp-E, PP1, and PNUTS Results antibodies were purchased from Bethyl Laboratories; histone PNUTS regulates the mitotic progression of mammalian cells H3, phospho-H3 Ser-10, phospho-Aurora A/B/C, Aurora B, and To investigate the expression level of PNUTS during the cell b-actin antibodies were purchased from Cell Signaling Technol- cycle, HeLa cells were released into cell-cycle progression from ogy; PP1 antibody was purchased from Santa Cruz Biotechnology. thymidine arrest. Interestingly, PNUTS expression is strongly PNUTS siRNA was obtained from Integrated DNA Technologies, elevated in mitosis (Fig. 1A), consistent with our previous finding with the targeting sequence of GGCGGCUACAAACUUCUU. in Xenopus egg extracts (35). Compared with cyclin B1, which PNUTS shRNA was generated using a pSUPER vector (Oligoengine), gradually accumulates prior to mitotic entry, PNUTS is more with the targeting sequence of CAGTGGTGGTTTCTGACAA. abruptly upregulated in M-phase (Fig. 1A). We then sought to examine the role of PNUTS in mammalian cell-cycle progression. Cell culture and treatment siRNA-mediated PNUTS knockdown significantly reduced the Human cervix carcinoma (HeLa) cells, authenticated by percentage of mitotic cells (Fig. 1B and C), suggesting a role of ATCC, were maintained in DMEM (Hyclone) with 10% FBS PNUTS in mitotic entry. Consistently, a synchronized mitotic (Hyclone). Breast cancer ZR75-1 and BT20 cells were authen- entry was not evident in cells treated with PNUTS siRNA, and then ticated at the University of Colorado Tissue Culture Core, and released from thymidine arrest (Fig. 1D). In addition to siRNA, the cultured in DMEM/F12 with 10% FBS. Cell-cycle synchroniza- expression of PNUTS was also suppressed by shRNA, which tion at G1–S was performed using two rounds of thymidine targeted a distinct sequence of PNUTS (Fig. 1E). Real-time micros- treatment (at a final concentration of 2 mmol/L). For mitotic copy revealed blocked mitotic entry in cells expressing PNUTS arrest, HeLa cells were treated with nocodazole (100 ng/mL) for shRNA (Fig. 1F). Moreover, although a smaller portion of cells 12 hours. Transfection was performed using Lipofectamine with PNUTS knockdown still entered mitosis, they exhibited high (Thermo Fisher Scientific), following a protocol recommended levels of chromosome misalignment in metaphase and chromatin by the manufacturer. bridges in anaphase (Fig. 2A and B). Collectively, these lines of

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Figure 1. PNUTS is upregulated in mitosis and plays an essential role in mammalian mitotic progression. A, PNUTS upregulation in mitotic cells. HeLa cells were synchronized by thymidine arrest, and then released for 0 to 8 hours. Cells were harvested and analyzed by immunoblotting. B, HeLa cells were treated with control or PNUTS siRNA, and then analyzed by immunoblotting. C, PNUTS knockdown reduced mitotic entry. HeLa cells with control or PNUTS siRNA were stained with DAPI and analyzed microscopically. At least 1,000 cells in multiple fields were examined. Mitotic cells exhibiting condensed chromosomes and nuclear envelope breakdown were morphologically identified. The percentage of mitotic cells in control or PNUTS siRNA–treated cells was quantified. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.05 was considered statistically significant (). D, HeLa cells were transfected with control or PNUTS siRNA. Thymidine release was performed as in A. Cells were harvested at the indicated time points, and analyzed by immunoblotting. The phosphorylation (band-shift) of Cdc27 indicates mitosis. E, HeLa cells were treated with PNUTS shRNA as described in Materials and Methods. The shRNA-expressing vector also contains a GFP-expressing cassette. As expected, the GFP-positive cell exhibits a lower level of PNUTS expression. F, PNUTS knockdown led to deficient mitotic entry. HeLa cells were treated with PNUTS shRNA as in E. These cells were synchronized by thymidine arrest and then released, as in A. Live cell imaging was performed as described in Materials and Methods to monitor mitotic progression. Representative images are shown. G, As in F, HeLa cells were released from thymidine arrest and analyzed by live cell imaging. Mitotic progression was monitored morphologically, as illustrated in F. The percentage of cells with mitotic entry within 10 hours is shown. At least 20 cells in multiple fields were examined. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.01 was considered statistically significant ().

evidence demonstrated an important role of PNUTS in mitotic human cell lysates confirmed PNUTS association with Aurora A, regulation. Aurora B, Cenp-E, Bub1, and PP1 at the endogenous level (Fig. 3B). Consistently, the reciprocal immunoprecipitation of Aurora PNUTS associates with Aurora kinases and kinetochore B recovered both PNUTS and PP1 (Fig. 3C). components We speculated that PNUTS modulates the action of PP1 toward PNUTS modulates PP1-dependent dephosphorylation of a subset of mitotic substrates. Thus, to reveal mechanistic insights Aurora A/B into the specific function of PNUTS, we used mass spectrometry to Identification of Aurora A and B complexes as associated identify proteins that copurified with PNUTS. Interestingly, the proteins of PNUTS prompted us to examine the potential role analysis identified Aurora A/Tpx2, Aurora B/Incenp complexes, of PNUTS in regulation of Aurora kinases. It has been shown that and a number of kinetochore proteins, including Cenp-E and PP1 antagonizes the functions of both Aurora A and Aurora B (14, Bub1 (Fig. 3A; Supplementary Table S1). Immunoprecipitation in 15, 22, 38). Interestingly, PNUTS blocked the PP1-dependent

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Figure 2. PNUTS knockdown disrupts mitotic chromosome segregation. HeLa cells were treated with control or PNUTS siRNA for 24 hours, and analyzed microscopically for chromosome misalignment in metaphase (A)and anaphase bridge (B). The percentages of mitotic cells (N > 50) exhibiting these defects, and representative images (with DAPI stain) are shown. Misaligned chromosomes and anaphase bridges are marked by arrow heads. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.01 was considered statistically significant (). dephosphorylation of Aurora B kinase in a reconstitutive phos- PNUTS depletion disrupts the mitotic localization of the phatase assay (Fig. 4A and B). A similar impact of PNUTS on PP1- chromosomal passenger complex mediated dephosphorylation of Aurora A kinase was also Upon metaphase-to-anaphase transition, the Aurora B– observed (Fig. 4C). Moreover, the collection of mitotic cells with containing chromosomal passenger complex (CPC) relocates PNUTS knockdown exhibited lower levels of Aurora A and B from anaphase chromosomes to the cell equator where it med- activation, as judged by autophosphorylation (Fig. 4D). The iates mitotic exit (3, 4, 40). Interestingly, we observed altered deficient Aurora phosphorylation was rescued with reexpression localization of Aurora B in anaphase with PNUTS knockdown: in of WT, but not W401A, PNUTS (Supplementary Fig. S1). Because anaphase cells with PNUTS knockdown, Aurora B was trapped on the W401A mutation disrupts the PNUTS–PP1 association (24, chromosomes rather than being released to spindles at the cell 30, 35), our findings indicate that PNUTS modulates Aurora equator (Fig. 5C). The dysregulation of Aurora B localization kinases in a PP1-dependent manner. Furthermore, immunoflu- correlated with mitotic chromosome missegregation after PNUTS orescent analysis of individual mitotic cells showed those that silencing (Fig. 5D). Moreover, a similar defect of INCENP local- exhibited metaphase and anaphase defects due to PNUTS knock- ization was observed in PNUTS-depleted cells (Fig. 5E). down lacked efficient phosphorylation of Aurora A/B (Fig. 5A). Histone H3 Ser-10 is hyperphosphorylated in mitosis, mediated The spatiotemporal regulation of PNUTS in mitotic progression primarily by Aurora B (39). We observed that PNUTS depletion The role of PNUTS in mitosis prompted us to ask how PNUTS significantly lowered the level of H3 Ser-10 phosphorylation in itself is spatiotemporally modulated. As expected, PNUTS is metaphase and anaphase (Fig. 5B). exclusively localized in the cell nucleus in interphase. From late

Figure 3. PNUTS associates with multiple mitotic regulators. A, As described in Materials and Methods, PNUTS pull-down was performed in Xenopus egg extracts that are particularly amenable for the biochemical isolation of large protein complexes, and analyzed by mass spectrometry to identify binding partners of PNUTS. The selected mitotic proteins and the number of identified peptides are shown. B, PNUTS immunoprecipitation (IP) was performed in HeLa cell lysates and analyzed by immunoblotting to confirm its association with Aurora A/B, Bub1, Cenp-E, and PP1. A control immunoprecipitation was performed using blank beads, and a 20% input lysate was included. C, Aurora B immunoprecipitation was performed in HeLa cell lysates and analyzed by immunoblotting to confirm its association with PNUTS and PP1. A control immunoprecipitation was performed using blank beads, and a 20% input lysate was included.

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Figure 4. PNUTS suppresses PP1-dependent inactivation of Aurora kinases. A, PNUTS prevents PP1-dependent dephosphorylation of active Aurora B. An in vitro phosphatase assay was performed using purified PP1, Aurora B, and PNUTS, as indicated. Samples were taken at the indicated time points and immunoblotted, as indicated. B, The PP1 phosphatase assay was performed as in A. The level of Aurora B phosphorylation was quantified using NIH ImageJ, and normalized to that of PP1. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.01 was considered significant (). C, An in vitro phosphatase assay was performed using purified PP1, Aurora A, and PNUTS, as indicated. Samples were taken at the indicated time points and immunoblotted, as indicated. D, PNUTS knockdown reduced mitotic phosphorylation of Aurora A/B. HeLa cells with or without PNUTS siRNA were synchronized by thymidine block and released for 9 hours for mitotic entry. Mitotic cells were collected by shake-off and analyzed by immunoblotting.

prometaphase to early telophase, the majority of PNUTS is PNUTS is a potential drug target to overcome the cancer removed from chromatin, with a small portion resides on spin- resistance to Aurora inhibition dles and poles (Supplementary Fig. S2). The reaccumulation of An important lesson learned from existing genetic and cellular PNUTS on chromosomes begins in telophase (Supplementary studies is that, although Aurora kinases play important functions Fig. S2), suggesting a potential involvement of PNUTS in the in mitosis, depletion of Aurora kinases can be well tolerated via decondensation of mitotic chromosome. Moreover, consistent downregulation of the counteracting PP1 (16–19). As we discov- with the protein association of PNUTS with kinetochore proteins ered PNUTS as a PP1-modulator toward Aurora kinases and their Cenp-E and Bub1 (Fig. 3), a portion of PNUTS colocalizes with substrates, we speculated that PNUTS targeting would synergize kinetochore proteins in interphase cells, as marked by Cenp-E with Aurora inhibition in conferring cytotoxicity. Interestingly, we and ACA. However, PNUTS is delocalized from kinetochores in observed that, compared with the treatment of an Aurora inhib- metaphase (Fig. 6A and B), indicating the dynamic dissociation of itor, ZM447439, alone, the combination of PNUTS siRNA and PNUTS from kinetochores. ZM447439 more profoundly decreased the viability of breast cancer ZR75-1 cells (Fig. 7A). This breast cancer cell line was PNUTS depletion attenuates the activation of the spindle selected because it was previously shown to be resistant to Aurora checkpoint kinase inhibition (41). Consistently, PNUTS siRNA and The role of PNUTS in regulation of Aurora kinases, and the ZM447439 synergistically induce cell death (Fig. 7B). The syner- dynamic kinetochore-association of PNUTS suggest a role of gistic effect between PNUTS siRNA and ZM447439 was also PNUTS in kinetochore signaling and the activation of the spindle conﬁrmed in BT20, another breast cancer cell line, and HeLa assembly checkpoint. Interestingly, PNUTS depletion accelerated (Supplementary Fig. S3A and B). the mitotic progression from nuclear envelope breakdown (NEBD) to the onset of anaphase (Fig. 6C). Moreover, PNUTS siRNA impaired metaphase arrest after nocodazole treatment Discussion (Fig. 6D), and diminished the phosphorylation and kinetochore PNUTS modulates PP1 during mitotic progression localization of BubR1 (Fig. 6E and F). Together, these lines of An evolutionarily conserved role of PP1 in mitotic regulation evidence indicate a role of PNUTS in the activation of the spindle has been well established in yeast, Drosophila, Xenopus, and checkpoint. mammalian cells (14, 15, 42, 43). Mammalian PP1 exhibits

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Figure 5. PNUTS knockdown disrupts the mitotic activation and localization of Aurora kinases. A, HeLa cells with control or PNUTS siRNA were analyzed by immunofluorescence for the phosphorylation of Aurora kinases. Misaligned chromosomes and anaphase bridges are marked by arrow heads. B, HeLa cells with control or PNUTS siRNA were analyzed by immunofluorescence for the phosphorylation of H3 Ser-10. Misaligned chromosomes and anaphase bridges are marked by arrow heads. C, HeLa cells with control or PNUTS siRNA were analyzed by immunofluorescence for Aurora B. Misaligned chromosomes and anaphase bridges are marked by arrow heads. D, As in C, 20 anaphase cells with either control or PNUTS knockdown were analyzed by immunofluorescence for Aurora B localization at either anaphase cell equator or anaphase chromosomes. Within the control and PNUTS siRNA–treated groups, cells were further classified into subgroups that exhibited either normal anaphase morphology or anaphase bridges. E, HeLa cells with control or PNUTS siRNA were analyzed by immunofluorescence for Incenp. Misaligned chromosomes and anaphase bridges are marked by arrow heads.

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Figure 6. PNUTS colocalizes with kinetochores in a cell cycle–dependent manner, and is required for the activation of the spindle checkpoint. A, HeLa were analyzed by immunofluorescence for PNUTS and Cenp-E. Representative images of cells in interphase and mitosis (metaphase) are shown. B, HeLa were analyzed by immunofluorescence for PNUTS and ACA (anticentromere antibody). Representative images of cells in interphase and mitosis (metaphase) are shown. C, HeLa cells with or without PNUTS knockdown were analyzed by live-cell imaging for mitotic progression. The time from NEBD to anaphase onset was documented. The mean values and SD are shown (N ¼ 20). Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.05 was considered statistically significant (). D, HeLa cells were treated with nocodazole for 12 hours and analyzed with light microscope for mitotic index, as in Fig. 1C. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. P < 0.001 was considered highly significant (). E, PNUTS knockdown disrupts BubR1 phosphorylation induced by nocodazole. HeLa cells were treated with nocodazole and PNUTS siRNA as indicated, and analyzed by immunoblotting. F, PNUTS knockdown reduces the kinetochore localization of BubR1. HeLa cells with or without PNUTS siRNA were treated with nocodazole and analyzed by immunofluorescence for BubR1. Representative images of metaphase cells are shown.

dynamic and isoform-specific localization at kinetochores, cen- Interestingly, mitotic regulation of PP1 involves a number of trosomes, chromosomes, and midbodies. Functionally, PP1 mechanisms that negatively modulate PP1 activities. For example, modulates the cell-cycle transitions of mitotic entry and exit, and PP1 is mitotically phosphorylated by CDK1, resulting in suppres- controls various aspects of mitotic progression, including nuclear sion of PP1 activity (48). Moreover, a specific inhibitor of PP1, envelope assembly, kinetochore signaling and spindle check- inhibitor-1, binds PP1 during mitosis to reinforce PP1 inhibition point, chromosome architecture, and cytokinesis (14, 15, 42, (49). Another PP1 inhibitor, inhibitor-2, modulates the action of 43). The complex functions of PP1 in mitosis rely on many PP1 toward multiple mitotic kinases, including Nek2A, Aurora, A targeting subunits that direct PP1 to specific mitotic substrates. and Aurora B (18, 50, 51). Our previous study in Xenopus egg For example, Repo-Man, KNL1, SKA, AKAP149, and Ki67 bind extracts suggested a role of PNUTS in the mitotic modulation of PP1, and recruit PP1 to anaphase chromosome, kinetochore, PP1 (35). In this study, we first confirmed the role of PNUTS in nuclear lamins, and other structures to govern mitotic progression mammalian mitosis: PNUTS expression oscillates during the cell (19, 43–47). cycle and peaks in mitosis; PNUTS knockdown reduced mitotic

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Figure 7. PNUTS targeting renders ZR75-1 breast cancer cells sensitive to Aurora inhibition. (A) ZR75-1 cells were treated with PNUTS siRNA and ZM447439 as indicated. The relative cell number (actual cell number/the starting cell number in day 1) is shown. The mean value and standard deviation were calculated from 3 independent experiments. Statistical significance was analyzed using one-way ANOVA and Turkey post hoc test. P < 0.001 was considered statistically significant (). (B) ZR75-1 cells were treated with PNUTS siRNA and ZM447439, as indicated, for two days, and measured by the trypan blue exclusion assay for cell death. The mean value and standard deviation were calculated from 3 independent experiments. Statistical significance was analyzed using an unpaired 2-tailed Student t-test. P < 0.001 was considered highly significant (). entry, and induced severe chromosome missegregation. We then additional molecular details are needed to further delineate this identified Aurora A, Aurora B-containing complexes, and several model. kinetochore proteins as specific binding patterns of PNUTS. PNUTS depletion suppressed the activation of Aurora A and PNUTS mediates kinetochore signaling and spindle checkpoint Aurora B kinases, inhibited the relocalization of the Aurora B– activation containing chromosomal passenger complex from anaphase Our study revealed PNUTS association with kinetochore pro- chromosomes to the cell equator, and bypassed the activation teins, Cenp-E and Bub1. A portion of PNUTS colocalizes with of the spindle checkpoint. Thus, our findings revealed a new Cenp-E and kinetochores in interphase, whereas PNUTS is exclud- mechanism involved in the inhibitory regulation of mitotic PP1. ed from kinetochores and chromosomes in metaphase. The In particular, this mechanism is responsible for the efficient dynamic kinetochore association and dissociation of PNUTS is activation and function of Aurora kinases in mitotic progression. potentially interesting. It is well recognized that reversible phos- Although the experiments were primarily carried out in HeLa cells, phorylation of kinetochore components plays a pivotal role in owing to the high efficiency of siRNA transfection in these cells, a governing spindle attachment and the activation/deactivation of portion of the studies were confirmed in additional breast cancer the spindle checkpoint. Several kinetochore components, espe- cell lines. Moreover, the role of PNUTS in mitotic progression was cially Knl1, yield PP1-binding activities, and were reported to first revealed in Xenopus egg extracts (35), indicating the well- recruit PP1 to kinetochores (19). After the proper spindle– conserved nature of this regulatory mechanism. kinetochore attachment, PP1 dephosphorylates Aurora B, Knl1, It remains a very intriguing question why the cell employs BubR1, and other kinetochore proteins to silence the spindle multiple distinct mechanisms to antagonize the activity of PP1 checkpoint and trigger metaphase–anaphase transition (22). This in mitosis. We speculate that the complex pattern of PP1 switch-like, abrupt activation of PP1 may involve mechanisms modulation is necessary to achieve the proper regulation of that prevent the premature action of PP1. Thus, it is plausible that PP1 during mitotic progression. On one hand, the bulk of PP1 PNUTS plays a role in suppressing the premature activation of activity needs to be inhibited to allow the phosphorylation of PP1, and that the kinetochore dissociation of PNUTS allows the mitotic substrates, which would otherwise be dephosphory- timely activation of PP1 on kinetochores, which then triggers lated by PP1. To this end, the antimitotic function of PP1 has metaphase–anaphase transition. Further investigations are need- been well established as an essential mechanism that promotes ed to better delineate the cell cycle–dependent kinetochore local- mitotic exit (42, 49). As an example, recent studies discovered a ization of PNUTS, as well as the detailed function of PNUTS in role of PP1 in the dephosphorylation of MASTL (also known as kinetochore signaling. Greatwall kinase) during mitotic exit (36, 52–54). On the other hand, portions of PP1 bind specificmitoticstructuresand PNUTS is a potential drug target to overcome the resistance to substrates, and play an active role in promoting several key Aurora kinase inhibition steps of mitotic progression. For example, it has been shown Small-molecule inhibitors of Aurora kinases have been sug- that mitotic PP1 governs the proper microtubule–kinetochore gested as promising therapeutics for breast, ovarian, lung, and attachment, silences the spindle checkpoint, and maintains the colon cancers, as well as hematologic malignancies (6, 8). Unfor- proper phosphorylation gradient and chromosome architecture tunately, the clinical benefits of these therapeutic agents are often (15, 42, 43). Interestingly, general inhibition of mitotic PP1 via limited by drug resistance. Therefore, revealing mechanisms that overexpression of nuclear inhibitor of PP1 (NIPP1) in HeLa allow cells to tolerate Aurora kinase inhibition is a crucial step cells caused prometaphase arrest, spindle-formation and chro- toward development of strategies that overcome drug resistance. mosome-congression defects, and hyperactivation of the spin- Interestingly, existing genetic and cellular studies showed that, dle checkpoint (55). In principle, the involvement of multiple although Aurora kinases play important functions in mitosis, inhibitory mechanisms allows for the dynamic, localization- depletion of Aurora kinases can be almost completely tolerated dependent, and substrate-specific modulation of PP1, although via downregulation of the counteracting PP1 (16–19). As we

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Wang et al.

discovered PNUTS as a PP1-modulator toward Aurora kinases and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, substrates, we speculated that that mitotic modulation of PP1 by computational analysis): F. Wang, L. Wang, A. Peng PNUTS may contribute to the cellular resistance to Aurora inhi- Writing, review, and/or revision of the manuscript: F. Wang, W. Wang, A. Peng fi Administrative, technical, or material support (i.e., reporting or organizing bition. We con rmed that PNUTS depletion sensitized resistant data, constructing databases): C. Li breast cancer cells to Aurora inhibition. Thus, it is plausible that Study supervision: C. Li, W. Wang, A. Peng PNUTS upregulation constitutes a mechanism of cancer resistance to Aurora inhibition. As such, PNUTS expression may serve as a Acknowledgments prognostic indicator for Aurora resistance, and targeting PNUTS Microscopic analysis was performed at the UNMC Advanced Microscopy expression or its PP1 interaction, in conjunction with Aurora Core Facility, supported by the Nebraska Research Initiative, the Fred and inhibition, may expand the therapeutic window, particularly in Pamela Buffett Cancer Center support grant (P30CA036727), and an Institu- cancer cells that respond poorly to the current clinical inhibitors of tional Development Award (IDeA) from the National Institute of General Aurora kinases. Medical Sciences of the NIH (P30GM106397). This work was supported by NIH grant R01CA172574 to A. Peng. Disclosure of Potential Conflicts of Interest The costs of publication of this article were defrayed in part by the payment of No potential conflicts of interest were disclosed. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: F. Wang, A. Peng Acquisition of data (provided animals, acquired and managed patients, Received November 15, 2017; revised May 31, 2018; accepted August 27, provided facilities, etc.): F. Wang, L. Wang, L.A. Fisher 2018; published first September 6, 2018.

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www.aacrjournals.org Mol Cancer Res; 17(1) January 2019 19

Phosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates Aurora Kinases and Mitotic Progression

Feifei Wang, Ling Wang, Laura A. Fisher, et al.

Mol Cancer Res 2019;17:10-19. Published OnlineFirst September 6, 2018.

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