Autophagy Governs Protumorigenic Effects of Mitotic Slippage–Induced Senescence Rekha Jakhar1, Monique N.H

Published OnlineFirst July 23, 2018; DOI: 10.1158/1541-7786.MCR-18-0024

Cell Cycle and Senescence Molecular Cancer Research Autophagy Governs Protumorigenic Effects of Mitotic Slippage–induced Senescence Rekha Jakhar1, Monique N.H. Luijten1, Alex X.F. Wong1, Bing Cheng1, Ke Guo1, Suat P. Neo2, Bijin Au3, Madhura Kulkarni1, Kah J. Lim4, Jiamila Maimaiti4, Han C. Chong1, Elaine H. Lim5, Tee B.K. Tan6, Kong W. Ong6, Yirong Sim6, Jill S.L. Wong7, James B.K. Khoo7, Juliana T.S. Ho7, Boon T. Chua8, Indrajit Sinha4, Xiaomeng Wang1,9, John E. Connolly3,10,11, Jayantha Gunaratne2,12, and Karen C. Crasta1,13,14,15

Abstract

The most commonly utilized class of chemotherapeutic hagy. Autophagy induction during mitotic slippage involved agents administered as a ﬁrst-line therapy are antimitotic the autophagy activator AMPK and endoplasmic reticulum drugs; however, their clinical success is often impeded stress response protein PERK. Pharmacologic inhibition of by chemoresistance and disease relapse. Hence, a better under- autophagy or silencing of autophagy-related ATG5 led to a standing of the cellular pathways underlying escape from cell bypass of G1 arrest senescence, reduced SASP-associated para- death is critical. Mitotic slippage describes the cellular process crine tumorigenic effects, and increased DNA damage after S- where cells exit antimitotic drug-enforced mitotic arrest and phase entry with a concomitant increase in apoptosis. Con- "slip" into interphase without proper chromosome segrega- sistent with this, the autophagy inhibitor chloroquine and tion and cytokinesis. The current report explores the cell fate microtubule-stabilizing drug paclitaxel synergistically inhib- consequence following mitotic slippage and assesses a major ited tumor growth in mice. Sensitivity to this combinatorial outcome following treatment with many chemotherapies, treatment was dependent on p53 status, an important factor to therapy-induced senescence. It was found that cells postslip- consider before treatment. page entered senescence and could impart the senescence- associated secretory phenotype (SASP). SASP factor produc- Implications: Clinical regimens targeting senescence and tion elicited paracrine protumorigenic effects, such as migra- SASP could provide a potential effective combinatorial strat- tion, invasion, and vascularization. Both senescence and SASP egy with antimitotic drugs. Mol Cancer Res; 16(11); 1625–40. factor development were found to be dependent on autop- 2018 AACR.

Introduction tubulin mutations and drug efﬂux pumps, and have yet to yield Antimitotic drugs are used extensively in the treatment of a improved outcomes for patients. Hence, a better understanding of variety of malignancies (1). The most commonly utilized class of possible cellular pathways and alternative molecular mechanisms antimitotic drugs are the microtubule poisons. These drugs inter- underlying escape from cell death are critical to address tumor fere with cellular proliferation by disrupting microtubules (MT), progression following MT-targeting drug treatment. thereby inducing a mitotic arrest that culminates in mitotic cell An alternative outcome to MCD following prolonged mitotic death (MCD; ref. 2). Unfortunately, the clinical success of arrest is mitotic slippage. Here, cells prematurely exit from MT-targeting drugs is often impeded by chemoresistance and mitosis and enter a pseudo-G1 phase without proper chromo- disease relapse. Thus far, the majority of studies addressing some segregation and cytokinesis. Postslippage cells are often mechanisms underlying therapy resistance have focused on characterized as tetraploid multinucleated cells due to nuclear

1Lee Kong Chian School of Medicine, Nanyang Technological University, Singa- and Cell Biology, Agency for Science, Technology, and Research, Singapore. pore. 2Quantitative Proteomics Group, Institute of Molecular and Cell Biology, 15Department of Medicine, Imperial College London, London, United Kingdom. Agency for Science, Technology, and Research, Singapore. 3Translational Note: Supplementary data for this article are available at Molecular Cancer Immunology Group, Institute of Molecular and Cell Biology, Agency for Science, Research Online (http://mcr.aacrjournals.org/). Technology, and Research, Singapore. 4Acenzia Inc, Windsor, Ontario, Canada. 5Division of Medical Oncology, National Cancer Centre Singapore, Singapore. R. Jakhar, M.N.H. Luijten, and A.X.F. Wong contributed equally to this article. 6Division of Surgical Oncology, National Cancer Centre Singapore, Singapore. Current address for M. Kulkarni: Transnational Cancer Research Centre, 7Division of Oncologic Imaging, National Cancer Centre Singapore, Singapore. Prashanti Cancer Care Mission, Pune and Indian Institute of Science Education 8Singapore Oncogenome Program, Institute of Molecular and Cell Biology, and Research, Pune, India. Agency for Science, Technology, and Research, Singapore. 9Vascular Biology Group, Institute of Molecular and Cell Biology, Agency for Science, Technology, Corresponding Author: Karen C. Crasta, Nanyang Technological University, Lee and Research, Singapore. 10Department of Microbiology and Immunology, Yong Kong Chian School of Medicine, 59 Nanyang Drive, Experimental Medicine Loo Lin School of Medicine, National University of Singapore, Singapore. Building, #04-12-01, Singapore 636921. Phone: 656-592-7870; Fax: 656-339- 11Institute of Biomedical Studies, Baylor University, Waco, Texas. 12Department 2889; E-mail: [email protected] of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, doi: 10.1158/1541-7786.MCR-18-0024 Singapore. 13School of Biological Sciences, Nanyang Technological University, Singapore. 14Genomic Instability and Cancer Group, Institute of Molecular 2018 American Association for Cancer Research.

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envelope assembly around condensed scattered chromosomes exception of hTERT-RPE-1 (DMEM-F12). Cells were freshly (3). Cells tend to undergo mitotic slippage rather than MCD thawed monthly and Mycoplasma testing was performed using when cyclin B1 degradation precedes proapoptotic signal accu- EZ-PCR Mycoplasma Test Kit (Biological Industries). Reagents mulation during prolonged mitotic arrest, as posited by the used in this study are included as Supplementary Tables S2 prevailing "competing networks-threshold" model (4). There and S3. can be several cell fates following mitotic slippage. One possible outcome is cell death postslippage, that fulfils the cyto- siRNA transfection toxic goal of therapy in addition to MCD (5). Cells can also Cells were transfected with siRNA using Lipofectamine RNAi- continue to proliferate as genomically unstable cells (5), there- MAX (Invitrogen) according to manufacturer's instructions. Cells by constituting a potential source of chemoresistance. In addi- were treated with drugs for 16 hours after transfection. siRNA tion, cells have been shown to arrest in the next interphase targeting scrambled sequence (#D-001810-10-05) and ATG5 postslippage and eventually enter cellular senescence (5). (#M-004374-04) were purchased from Dharmacon and used Indeed, cellular senescence has emerged as a major outcome according to the manufacturer's instructions. of a variety of chemotherapies in a process known as therapy- induced senescence (6). Senescence-associated b-galactosidase staining Classically, senescence is considered to be a barrier against Cells were stained using the senescence b-galactosidase tumorigenesis, as it restricts cell proliferation (7). This is mediated (SA-b-gal) Staining Kit (#9860; Cell Signaling Technology) by the senescent cell secretome, known as the senescence-associ- according to manufacturer's instructions. ated secretory phenotype (SASP), which consists of a variety of cytokines, chemokines, growth factors, and matrix metallopro- BrdU incorporation assay teases (8). In addition to reinforcing stable growth arrest via both Cells were incubated with BrdU (BrdU Labeling and Detection autocrine and paracrine signaling, SASP factors also promote Kit, Roche) for 16 hours and visualized according to the manu- immunosurveillance of senescent cells, leading to tumor remis- facturer's instructions. sion (9). In this way, senescence serves as an important tumor- suppressive mechanism. However, senescent cells also possess Immunoblotting oncogenic potential via paracrine effects of the SASP. SASP factors Cells were lysed in RIPA buffer (Pierce) containing Protease/ have been shown to engender protumorigenic effects such as Phosphatase Inhibitor Cocktail (Thermo Scientific) and equal cellular motility (invasion, migration, and metastasis), epitheli- amounts of total protein were subjected to SDS-PAGE. al–mesenchymal transition (EMT), proliferation, angiogenesis, as well as inflammation in neighboring cells (8). Flow cytometry Molecular mechanisms underlying senescence following After fixation in ice-cold 70% ethanol overnight, cells were mitotic slippage and their consequential cell fate significance stained with propidium iodide, and incubated with phospho- for MT-targeting therapies have not been well-explored. Here, Histone H3 antibody conjugated to Alexa Fluor 488 (Cell we examine the senescent cell fate following nocodazole Signaling Technology). Samples were analyzed using Accuri C6 (MT-destabilizing agent) or paclitaxel (MT-stabilizing agent)- flow cytometer (BD Biosciences). induced mitotic slippage. Our results demonstrate that mitotic slippage–induced senescence could confer paracrine tumori- Immunofluorescence genic effects via the SASP. We sought to improve therapeutic Cells were fixed in 4% paraformaldehyde in PBS for 10 minutes outcomes and identified ER stress–triggered autophagy as an at room temperature. Antibody incubation and staining were effector of mitotic slippage–induced senescence. We found performed as described previously (10). that inhibition of autophagy via pharmacologic means or silencing of autophagy-associated genes could override senes- Live-cell imaging cence, leading to cell death upon antimitotic drug treatment. Cells were imaged on a Nikon inverted fluorescence micro- Importantly, combination treatment of tumors in mice with scope at multiple sites every 30 minutes for 48–72 hours using a often-used paclitaxel and autophagy inhibitor chloroquine Plan Apo 40 objective. showed significant tumor growth arrest, underscoring autophagic inhibition as a potential strategy of tackling resistance. Preparation of conditioned media We further found that xenograft tumors with wild-type p53 Cells were treated with drugs for two days followed by culture in exhibited a superior response toward combinatorial therapy drug-free media containing 0.5% FBS for additional two days. compared with tumors with p53 knockout. This suggests that Culture media were then centrifuged at 5,000 g, filtered through sensitivity toward this treatment is dictated by p53 status, 0.22-mm pore filter (Pall Corporation) and mixed with media which could serve as a potential biomarker in predicting containing 40% FBS in a proportion of 3:1 to generate condi- clinical response. tioned media (CM) containing 10% FBS.

Clonogenic assay Materials and Methods Cells were treated with drugs for 72 hours and reseeded at Cell culture and reagents 5,000 cells per 6-well, followed by culture for 10 days with fresh U2OS, HCT116, MCF7, MDA-MB-231, PanC1, MIA PaCa2, media supplemented every other day. Colonies were stained HEK293T, and hTERT-RPE-1 cells were purchased from ATCC with 0.05% crystal violet (Sigma). Number of colonies was at the start of the project in 2014. All cell lines were cultured in analyzed using GelCount (Oxford Optronix) according to DMEM supplemented with 10% FBS (Hyclone GE), with the manufacturer's instructions.

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Cell viability assay Nanyang Technological University Institutional Review Board Cells were seeded at 4,000 cells per 96-well and treated with (IRB-2018-04-009). Written informed consents were obtained drugs for 72 hours. Cell viability was assessed using CellTiter from participating patients. Biopsies were collected using core 96 One Solution Proliferation Assays Kit (G3580; Promega) needle biopsy procedure and were snap-frozen in liquid nitrogen according to manufacturer's instructions. immediately after aspiration. Frozen biopsies were thawed slow- ly, fixed, and processed accordingly for IHC. RNA extraction and qRT-PCR Total RNA was extracted using RNeasy plus Mini Kit (QIAGEN) IHC according to manufacturer's instructions. cDNA was reverse tran- After fixation, sections were incubated with primary antibodies scribed using iScript RT Supermix (Bio-Rad) and subjected to overnight at 4C, followed by incubation with biotinlyated sec- SYBR Kit (#4472942; Life Technologies). Relative expression ondary antibodies (Vector Laboratories). Sections were then values of each gene were normalized to GAPDH expression. incubated in Avidin:Biotinylated Enzyme Complex (ABC; Vector Primers used are in Supplementary Table S4. Laboratories) for 30 minutes, developed with 3,30-diaminoben- zidine (DAB) substrate (Vector Laboratories), and nuclei counter- Multiplex cytokine analysis stained with hematoxylin. Slides were mounted with Fluka Eukitt Forty-one analytes from Human Cytokine Panel 1 (Merck quick-hardening mounting medium (Sigma-Aldrich). Millipore) were measured as per manufacturer's instructions. Plates were washed using Tecan Hydrospeed Washer (Tecan) and SILAC labeling read with Flexmap 3D system (Luminex Corp). Data were ana- RPE-1 cells were cultured in SILAC DMEM/F12 media, con- lyzed using Bio-Plex manager 6.0 (Bio-Rad) with a 5-parameter taining light or heavy arginine and lysine ["R0K0 DMEM/F12" 12 12 curve-fitting algorithm applied for standard curve calculations. with C6-L-arginine (Sigma-Aldrich) and C6-L-lysine (Sigma- 13 Aldrich) or "R10K8 DMEM/F12" with C6-L-arginine 13 Tumorigenic phenotypic assays (Cambridge Isotope) and C6-L-lysine(CambridgeIsotope)] For scratch wound migration assay, cells were incubated with and supplemented with dialyzed FBS. SILAC labeling was CM for two days. At 90% confluency, the cell monolayer was performed as described previously (11). After lysis in RIPA scratched and the wound closure rate tracked. For cell invasion buffer (Thermo Scientific) with Protease/Phosphatase Inhibitor assay, cells expressing H2B-GFP were incubated with CM for two Cocktail (Thermo Scientific), equal amounts of protein from days, plated on top surface of transwell filter chambers precoated R0K0andR10K8weremixedandboiledwithloadingbufferat or uncoated with Matrigel (BD Biosciences), and the percentage of 95C for 5 minutes. Protein complexes were separated by one- invasive cells determined after 24 hours. For choroid angiogenesis dimensional 4%–12% NuPage Novex Bis–Tris Gel (Invitrogen), assay, segments of the peripheral choroid layer from eyes of P3 stained with Colloidal Blue Staining Kit (Invitrogen), and sub- mice were incubated with CM 1:3 diluted with EGM2 media jected to in-gel digestion as described previously (11). (Lonza) over four days and imaged under phase contrast. Additional Materials and Methods can be found under Sup- plementary Methods. Quantification of cellular migration and metastasis in zebrafish (ZgraftTM) U2OS H2B-GFP cells were incubated with CM for two days Results and stained with DiI (Vybrant, Life Technologies) before injec- Postslippage tetraploid cells can enter senescence tion into zebrafish embryos. Embryos were imaged 48 hours To study the fate of cells postslippage, we chose mitotic slip- after injection to determine the metastatic tumor foci position page–prone cells, namely osteosarcoma U2OS cells, colorectal relative to injection site. carcinoma HCT116 cells, and nontransformed telomerase- immortalized hTERT-RPE-1 cells (hereafter referred to as RPE-1). Human tumor xenografts Mitotic slippage, instead of MCD, was previously found to be the All animal studies were approved by the Institutional Animal preferred pathway in these cells upon treatment with MT poisons Care and Use Committee (IACUC no. 151103) of the Biological (4). To achieve maximum homogeneity in the cell population, Resource Centre and were carried out under the policies from the U2OS and RPE-1 cells were first synchronized before treatment Animal Facility Centre of the Agency for Science, Technology and with the microtubule-depolymerizing drug, nocodazole for a Research (ASTAR, Singapore). HCT116 colon cancer cells were prolonged period of 72 hours to allow cells to undergo mitotic injected subcutaneously into both dorsal flanks of female BALB/c slippage (experimental scheme in Supplementary Fig. S1A). Cells athymic nude mice (nu/nu) (InVivos; n ¼ 5 mice in each treat- were monitored by time-lapse microscopy from the start of ment group). When tumor volume reached around 300 mm3, nocodazole treatment (t ¼ 0 hours). U2OS and RPE-1 cells mice were injected intraperitoneally with respective treatment predominantly displayed a "rounded up" morphology, typical every three days for two weeks. Tumor volume was measured of mitotic arrest, at 16 hours post-nocodazole treatment (Sup- every three days from start of treatment. Mice were sacrificed at the plementary Fig. S1A). By 72 hours post-nocodazole treatment, end of the treatment schedule and all tumors were harvested for 70.9% of U2OS cells and 87.8% of RPE-1 cells were observed to weight measurement, IHC, and immunofluorescence. have undergone blebbing and reflattening without cytokinesis (characteristics of mitotic slippage). This was further confirmed by Patient breast cancer tissues time-course experiments in synchronized nocodazole-treated This study was conducted in accordance with recognized ethical U2OS cells that showed decreased levels of mitotic cyclin B1, guidelines (Singapore Guideline for Good Clinical Practice, mitotic marker phosphorylated Histone H3, and spindle assem- Declaration of Helsinki and Belmont Report) and approved by bly checkpoint markers BubR1 and Bub1 by immunoblotting

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Figure 1. Postslippage cells elicit SASP that confers tumorigenic effects. A, Experimental scheme and representative images of U2OS and RPE-1 cells treated with nocodazole (Noc) for 0, 3, 6, and 9 days and stained for SA-b-gal. Plot shows percentage of SA-b-gal–positive cells. (Continued on the following page.)

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(Supplementary Fig. S1B). In addition, a mitigated decline in cells (Supplementary Fig. S2B). Interestingly, treatments that levels of antiapoptotic protein Mcl-1 and corresponding increase resulted in senescence, namely paclitaxel and ZM, showed high in cell death marker cleaved PARP was detected. An accumulation degrees of multinucleation, whereas Mon-treated cells were most- of multinucleated tetraploid G1 U2OS cells was also observed, as ly mononucleated (Supplementary Fig. S2C). This could imply shown by 4C-G1 population by flow cytometry and multiple that postslippage multinucleated cells may have enhanced pro- nuclei by phase-contrast microscopy (Supplementary Fig. S1C clivity to enter senescence. Indeed, a previous report demonstrat- and S1D). A similar increase in multinucleation was seen in ed that tetraploid cells with irregular-shaped nuclei progressively nocodazole-treated HCT116 cells postslippage (Supplementary developed senescence following Aurora kinase B inhibition (16). Fig. S1E). To determine whether mitotic slippage–induced senescence Because cells following prolonged nocodazole treatment dis- could be observed in vivo, we treated HCT116 xenograft mice played an enlarged, flattened morphology reminiscent of senes- with either nocodazole or paclitaxel. Hematoxylin and eosin cence, we stained cells for the SA-b-gal. As shown in Fig. 1A, at day staining of tissue sections revealed a discernible increase in 3 post-nocodazole treatment, about 45% of U2OS cells and 70% multinucleated cells in the nocodazole- or paclitaxel-treated of RPE-1 cells stained positive for SA-b-gal, indicating entry into xenografts compared with vehicle-treated control (Supplementa- senescence. Negligible BrdU staining in multinucleated cells ry Fig. S3A). In addition, the majority of cells that stained positive compared with control indicating a lack of cell proliferation for SA-b-gal were observed to be multinucleated (Supplementary further supporting this finding (Supplementary Fig. S1F). To Fig. S3A), suggesting possible correlation between multinuclea- confirm this as a bona fide senescence phenotype, nocodazole tion and senescence. Nocodazole- or paclitaxel-treated xenografts was removed and cells were cultured in fresh media for an also showed a significant increase in both p21 mRNA and protein additional 6 days (experimental scheme in Fig. 1A). A progressive levels compared with vehicle-treated control (Supplementary increase in the number of cells that entered senescence was Fig. S3B and S3C). In addition, tissue biopsies from invasive observed (Fig. 1A), confirming that cells underwent a stable ductal breast carcinoma from patients treated with paclitaxel cell-cycle arrest. Loss of lamin B1 has been associated with showed increased populations of cells in the humoral region that senescence (12). Our data show a decrease in lamin B1 levels stained positive for p21 expression as compared with biopsies after day 3 (Fig. 1B). Increased cyclin-dependent kinase inhibitor obtain from patients before treatment (Supplementary Fig. S3D). p21, tumor suppressors p53 and total retinoblastoma protein (pRb), and hypophosphorylation of pRb at Ser780 (13) com- Postslippage secretory factors facilitate paracrine tumorigenic pared with control further confirmed the senescence phenotype at phenotypes day 3 (Fig. 1B). Interestingly, we observed a decline in p53 levels SASP factors secreted by cells which have undergone senescence from day 6 onwards even though the senescence phenotype due to replicative exhaustion, oncogene activation, or irradiation persisted. This is consistent with a report showing downregulation can modulate the tissue microenvironment to stimulate tumor of p53 to be crucial for induction of SASP (14). By inference, progression (8). As a first step to ascertain potential tumorigenic because p21 is part of the p53–p21 senescence pathway where role for SASP following mitotic slippage, we performed gene p53 activates p21, this could also explain the corresponding expression microarray analysis on U2OS cells treated with either decrease observed for p21 (15). Taken together, our results DMSO (control) or nocodazole for 48 hours. Microarray data confirmed that cells postslippage entered senescence following confirmed SASP factor expression, including cytokines and che- G1 arrest. mokines IL1a, IL1b, CXCL8, and CCL3 among the upregulated To determine whether antimitotic drugs broadly induce senes- genes (Supplementary Fig. S4A). This was further confirmed at the cence, cells were treated with the MT-stabilizing drug paclitaxel protein level with stable isotope labeling with amino acids in cell (PTX), Aurora kinase B inhibitor ZM447439 (ZM), or kinesin- culture (SILAC) followed by high-resolution mass spectrometry related Eg5 inhibitor Monastrol (Mon). SA-b-gal staining showed (MS; Supplementary Fig. S4B; ref. 11). Notably, mass spectro- discernible differences in the extent of senescence, as paclitaxel metric analysis revealed upregulation of SASP factors including and ZM significantly increased the percentage of SA-b-gal– IL1b and IL8, and senescence-associated histone H3.3 variant positive cells while Mon-treated cells hardly displayed signs of (ref. 17; Supplementary Fig. S4C; Supplementary Table S1). senescence (Supplementary Fig. S2A). Increased SA-b-gal staining In addition, both quantitative real-time PCR (qRT-PCR) and was also observed for nocodazole- and paclitaxel-treated HCT116 Luminex assays confirmed upregulated expression and secretion

(Continued.) One-hundred cells per condition and data are mean SD of two independent experiments. Scale bar, 10 mm. B, Cell lysates collected as per A were subjected to immunoblotting (IB) and probed for senescence-associated markers. The signal intensity ratios are shown at the bottom of the relevant lanes. C, Left, qRT-PCR analysis shows relative mRNA expression of SASP components in DMSO-treated or nocodazole-treated U2OS cells for 72 hours. mRNA levels were normalized to GAPDH and DMSO control. Right, Plot shows relative fold change of cytokine secretion from U2OS cells treated with DMSO or nocodazole for 72 hours using Luminex analysis. Data are mean SD of three independent experiments. D, Scheme depicts the preparation of conditioned media (CM) for determining paracrine tumorigenic effects in Fig. 1E–H. E, U2OS and RPE-1 cells carrying chromosomal marker H2B-GFP were incubated with their respective CM for 2 days and subjected to the scratch wound healing assay. Left, Representative images of CM-treated U2OS H2B-GFP cells taken at 0 hours (immediately after scratching) and at the indicated time intervals. Right, Plot shows rate of wound closure in U2OS and RPE-1 cells carrying H2B-GFP. Data are mean SD of three independent experiments. Scale bar, 200 mm. F, U2OS H2B-GFP cells were treated as per E. Invasive ability was assessed by transwell Matrigel assay. Scale bar, 200 mm. Plot shows percentage of invasive cells. n ¼ 5 random ﬁelds were taken per condition and data are mean SD of three independent experiments. G, Cells collected from F were subjected to qRT-PCR. Plot shows the relative mRNA expression of migration-associated markers following incubation with indicated CM. mRNA levels were normalized to GAPDH and DMSO control. Data are mean SD of three independent experiments. H, Representative images of choroidal sprouts formed after culturing with indicated CM for 2 days. Plot shows choroid sprouting ratio (area of vascular sprouting/area of the explant). Minimum of 6 explants analyzed per treatment. Data are mean SD of three independent experiments. , P < 0.05; , P < 0.005; , P < 0.001; , P < 0.0001; ns, not signiﬁcant by Student t test.

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of various SASP factors in nocodazole-treated U2OS cells at adaptor protein p62/SQSTM1. To ensure that autophagy flux was 72 hours postslippage compared with DMSO-treated control assessed only in adherent postslippage cells destined to enter cells (Fig. 1C). senescence, and that quantitation was not affected by cell death The observed increase in SASP factor secretion prompted us to postslippage, cells were washed 36 hours post nocodazole test whether SASP factors could mediate tumorigenic phenotypes treatment to remove apoptotic cells. As shown by phosphorylated such as migration, invasion, and angiogenesis. To this end, we Histone H3 levels in Fig. 2B and C, HCT116 and U2OS cells were prepared conditioned media (CM) from RPE-1 and U2OS cells in mitosis (M) at t ¼ 16 hours and postslippage (PS) from t ¼ 24– treated with nocodazole for 48 hours for use in various pheno- 36 hours. Increased autophagy flux postslippage was observed as typic assays (experimental scheme in Fig. 1D). To assess migratory shown by an increase in LC3-II isoforms at t ¼ 24–36 hours capability, RPE-1 and U2OS cells expressing chromatin marker compared with t ¼ 16 hours (Fig. 2B and C). To confirm this, H2B-GFP exposed to CM from their respective postslippage U2OS cells were treated with chemical inhibitor of autophagy parental cells were subjected to the scratch wound healing assay. Bafilomycin A1 (Baf A1), which blocked lysosome acidification As shown in Fig. 1E, postslippage CM (nocodazole CM) promot- and prevented autophagosome clearance. Increased LC3-II was ed wound closure at a rate faster than control CM (DMSO CM), observed at t ¼ 36 and 48 hours (Fig. 2C) compared with indicating increased induction of migration. Notably, cell prolif- nocodazole control, suggesting LC3-II accumulation postslippage eration assays by BrdU labeling and cell count experiments to be due to autophagosome accumulation and not impairment demonstrated that factors secreted did not alter rate of cell of downstream autophagic processes such as autophagosome– proliferation in CM-exposed cells (Supplementary Fig. S5A and lysosome fusion or lysosomal degradation. Concomitantly, a S5B), indicating increased wound closure was not influenced by decrease in p62, which can be blocked by Baf A1, further con- proliferation. To evaluate invasive capability, U2OS H2B-GFP firmed active autophagy (Fig. 2B and C). LC3-II accumulation in cells incubated with either control or postslippage CM were used postslippage cells was also blocked by knockdown of autophagy in transwell invasion assays. An increased number of cells exposed by stable expression of short hairpin RNA targeting ATG5 to postslippage CM invaded the bottom of the filter chamber (shATG5) required for autophagy elongation (ref. 20; Fig. 2D). compared with control CM (Fig. 1F). An upregulation of invasion Autophagic induction occurs via a number of pathways that and migration-related markers fibronectin and MMP9 in U2OS finally converge on regulation of the AMPK/ULK/mTOR axis, cells exposed to postslippage CM compared with control CM was which integrates growth factor and nutrient signals to regulate also observed (Fig. 1G), confirming migratory and invasive capa- cellular metabolism and maintain energy homeostasis (21). bilities. To test for angiogenic capability, we used the choroid Phosphorylation of AMP-activated protein kinase (AMPK) on angiogenesis assay, an ex vivo model of angiogenesis. Increased Thr172 activates autophagy by directly activating Unc-51 Like induction of vascular sprouting from choroid explants incubated Autophagy Activating Kinase 1 (ULK1) through phosphorylation with postslippage CM compared with control CM was observed of Ser317 and Ser777 (21, 22). In contrast, high activity of the four days after incubation (Fig. 1H), demonstrating angiogenic mTOR negatively regulates autophagy by preventing ULK1 acti- capability conferred by factors from postslippage cells. Taken vation via ULK1 Ser757 phosphorylation and disruption of ULK1 together, we conclude that postslippage SASP proteins confer and AMPK interaction (21). To test whether cells postslippage paracrine protumorigenic potential. invoked the AMPK/ULK/mTOR axis for autophagy induction, nocodazole-treated U2OS cells were subjected to immunoblot- Autophagy activity increases following mitotic slippage ting. We observed that in mitotic cells 16 hours post-nocodazole We next wished to investigate the molecular determinants that treatment, both mTOR and AMPK were activated as shown by induce senescence in postslippage cells. Mass spectrophotometric increased phosphorylation of the mTOR substrate p70 S6K, analysis of U2OS cells treated with nocodazole for 48 hours, conversion of total ULK1 to phosphorylated Ser757 ULK1 as well revealed four autophagy-related proteins, namely MAP1A, as increased phosphorylated Thr172 AMPK (Fig. 2E). This corre- MAP1B, MAP1LC3B (also known as LC3B), and GABARAP to be lated with enhanced autophagy, and was consistent with an downregulated postslippage compared with control (Supplemen- observed increase in LC3-II (Fig. 2E). As it was previously reported tary Fig. S4C; Supplementary Table S1). Because all four of these that autophagy can be activated in a mTOR-independent manner proteins serve as autophagic substrates, their downregulation (23, 24), our findings suggest presence of autophagic activity suggests increased autophagic activity. Autophagy is a catabolic despite mTOR activation during mitosis. On the other hand, process in which cytoplasmic constituents are targeted for remov- diminished levels of phosphorylated-p70 S6k indicating al or recycling in autophagosomes that fuse with the lysosome decreased mTOR activity with concomitant decrease in ULK1 (18). MAP1A and MAP1B are microtubule-associated proteins, phosphorylated on Ser757 residue was observed in postslippage which are precursor polypeptides that undergo proteolytic pro- cells (Fig. 2E). These findings together with the observed increase cessing to generate heavy and light chain subunits such as for phosphorylated Thr172-AMPK and LC3-II levels, strongly MAP1LC3A and MAP1LC3B (19). MAP1LC3B and GABARAP suggested that the AMPK/ULK/mTOR axis promotes autophagy belong to the ATG8 orthologs of mammalian cells (19). Consis- induction postslippage. tent with upregulated autophagy postslippage, time-lapse microscopy revealed increased autophagic marker GFP-LC3 punctate AMPK activation is induced upon ER proteotoxic stress foci in U2OS cells postslippage (t ¼ 48 hours), indicating autop- postslippage hagosome accumulation (Fig. 2A). We next sought to understand the molecular mechanism by To further evaluate autophagic flux, we assessed conversion which mitotic slippage could lead to AMPK activation and of the nonlipidated form LC3B-I to the lipidated autophagosome- autophagy induction. A recent report showed that aneuploidy associated form LC3B-II (hereafter LC3-I and LC3-II, respectively), caused substantial endoplasmic reticulum (ER) proteotoxic stress as well as degradation of the ubiquitin-binding autophagic and unfolded protein response (UPR) activation in cells that

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Figure 2. Autophagy is induced via AMPK/ULK1/mTOR axis during mitotic slippage. A, U2OS cells stably expressing GFP-LC3 were assessed for GFP puncta formation following 48-hour nocodazole (Noc) treatment. Plot shows percentage of GFP-LC3 puncta-positive cells (>5 puncta per cell) following indicated treatments. Fifty cells per condition. Scale bar, 20 mm. B, HCT116 cells were treated with nocodazole for indicated duration. Lysates from mitotic cells (M) or postslippage cells (PS) were subjected to IB and probed for autophagy- and mitotic slippage–related markers. The respective signal intensity ratios are shown at the bottom of the relevant lanes. C, U2OS cells were treated with nocodazole for the indicated duration with or without autophagy inhibitor Baﬁlomycin A1 (Baf A1). Lysates from mitotic cells (M) or postslippage cells (PS) were subjected to IB analysis and probed for autophagy-related markers LC3 and p62. D, U2OS cells stably expressing shCtrl or shATG5 were treated with nocodazole for the indicated time and subjected to IB analysis. The LC3-II/GAPDH and p62/GAPDH signal intensity ratios are shown at the bottom of the relevant lanes. E, U2OS cells were treated with nocodazole for the indicated duration, subjected to IB and probed with AMPK/mTOR/ULK1 axis–related antibodies. The respective signal intensity ratios are shown at the bottom of the relevant lanes. All data are mean SD of three independent experiments (, P < 0.001 by Student t test). underwent cell death postslippage (25). We therefore examined ylates eukaryotic translation initiator factor 2a (eIF2a) on Ser51, the expression of UPR-related protein kinase RNA-like ER kinase which then attenuates global protein synthesis. Phosphorylated (PERK) and inositol-requiring protein 1 (IRE1) in U2OS cells eIF2a leads to an increase in transcription factor CCAAT/enhancer after nocodazole and paclitaxel treatment. Upon accumulation binding protein (C/EBP) homologous protein (CHOP) at both of misfolded or unfolded proteins in the ER, PERK is activated transcriptional and translational level. Our data revealed that through dimerisation and trans-autophosphorylation on multi- nocodazole and paclitaxel treatment increased the phosphoryla- ple residues including Thr980 (26). Activated PERK phosphor- tion of PERK at Thr980 and eIF2a at Ser51 postslippage compared

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Figure 3. ER stress mediates postslippage AMPK activation. A, U2OS cells were treated with nocodazole (Noc) or paclitaxel (PTX) for the indicated duration. Lysates from mitotic cells (M) or postslippage cells (PS) were subjected to IB and probed for ER stress–related markers. The respective signal intensity ratios are shown at the bottom of the relevant lanes. B and C, U2OS cells were treated with nocodazole or paclitaxel for the indicated time points. Cells were treated with 1 mg/mL tunicamycin (TM) for 24 hours as a positive control. Relative CHOP expression and splicing of XBP1 mRNA was detected by RT-PCR and quantiﬁed by qRT-PCR. D, U2OS cells were treated with nocodazole in absence or presence of 10 mmol/L PERK inhibitor (PERKi; GSK2656157), subjected to IB and probed for autophagy-related markers. E, U2OS cells were treated with nocodazole in absence or presence of 10 mmol/L IRE1 inhibitor (IRE1i; 4m8C), were collected at the indicated time points and analyzed by IB with the indicated antibodies. All data are mean SD of three independent experiments (, P < 0.05; , P < 0.005; , P < 0.001 by Student t test).

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with control (t ¼ 0 hours, start of treatment; Fig. 3A). An increase stress shown by increased RPA32 phosphorylation at 72 hours in CHOP at both protein and mRNA levels was also observed post-nocodazole treatment compared with control was also postslippage compared with control (Fig. 3A and B). An alterna- observed (Fig. 4I). Taken together, our findings indicate that tive signaling branch of the UPR is the IRE1–XBP1 pathway (27). autophagy modulates cell fate upon antimitotic drug treatment Phosphorylation of IRE1 leads to cleavage of XBP1 mRNA and and that inhibition of autophagy engenders cells to bypass generates a translational frameshift which acts as a potent tran- senescence postslippage and enter S phase. These cells then scriptional activator (26). Increased IRE1 phosphorylation at undergo increased replication stress and DNA damage, culminat- Ser724 and XBP1 mRNA splicing was observed postslippage ing in cell death. compared with DMSO-treated control (Fig. 3C). Thus, in addition A recent study reported that autophagy plays a role in promot- to cell death postslippage, our results indicate induction of ER ing MCD during prolonged mitotic arrest (30), suggesting that stress and UPR induction with activation of PERK and IRE1 UPR entry into mitotic slippage per se could potentially be inhibited by pathways in postslippage senescent cells as well. autophagy. We therefore tracked individual cell fate (MCD vs To investigate if either of these pathways regulate autophagy, mitotic slippage) in U2OS cells stably transfected with shATG5 PERK or IRE1 activity was inhibited with PERK inhibitor treated with either nocodazole or paclitaxel for 48 hours using GSK2656157 or IRE1 inhibitor 4m8C. GSK2656157 reduces both time-lapse microscopy. Intriguingly, there was no significant total and phosphorylated PERK levels, while 4m8C blocks sub- difference in the proportion of cells undergoing MCD or slippage strate access to the active site of IRE1 and selectively inactivates upon autophagic inhibition compared with control (Supplemen- both XBP1 splicing and IRE1-mediated mRNA degradation. Inter- tary Fig. S7A). Consistently, the duration from mitosis to mitotic estingly, while inhibition of IRE1 did not prevent the accumula- slippage (Supplementary Fig. S7B), cyclin B1 degradation, and tion of LC3-II, PERK inhibition decreased LC3-II levels in dephosphorylation of BubR1 and Histone H3 were not affected postslippage cells to that of cycling cells (Fig. 3D and E), revealing by autophagy inhibition postslippage (24–36 hours post- that autophagy induction postslippage occurred through PERK nocodazole treatment; Supplementary Fig. S7C and S7D). Our activation. As described previously (Fig. 2), autophagy was affect- results thus indicate that autophagy modulates cell fate specifi- ed via AMPK activation, which is downstream of PERK (28). We cally after escape from mitotic arrest through mitotic slippage. find that PERK inhibition led to decreased AMPK phosphorylation (Fig. 3D), further confirming that the increase in LC3-II was Autophagy inhibition attenuates protumorigenic effects predominantly induced by the PERK–AMPK axis of the ER stress of SASP response in postslippage cells. We observed a discernible decrease in expression of SASP cytokines CXCL3, IL1b, IL6, IL8, CCL7, and PDGF-AB/BB and Autophagy inhibition in postslippage cells leads to senescence increased expression of BMP2 postslippage following autophagy bypass and cell death inhibition compared with control (Fig. 5A; Supplementary Fig. Because autophagy is active postslippage, we sought to inves- S8A and S8B). This suggests that autophagy enables senescence tigate whether the senescence cell fate could be influenced and consequently modulates SASP following mitotic slippage. by autophagic inhibition. Inhibition of autophagy by Baf A1 or Notably, IL1b regulatory control via the autophagy axis was transfection with shATG5 resulted in senescence bypass as observed to be more prominent posttranscriptionally as shown observed by decreased SA-b-gal staining (Fig. 4A; Supplementary in Supplementary Fig. S8B (compare with mRNA level in Sup- Fig. S6A). Concomitantly, a substantial increase in cells entering S- plementary Fig. S8A). In addition, SASP-induced tumorigenic phase was suggested by increased BrdU labeling upon inhibition function was attenuated following autophagic inhibition (Fig. 5). of autophagy (Fig. 4B). Transfection of siATG5 clearly suppressed Postslippage CM promoted increased vascular sprouting com- the increase in LC3-II indicating autophagy was indeed blocked pared with control CM from cycling cells in choroid explants, (Fig. 4C). This unequivocally confirmed that postslippage senes- whereas autophagic inhibition significantly inhibited sprouting cence was dependent on autophagy. Time-lapse microscopy activity (Fig. 5B). In addition, transwell assays using cells incu- revealed that autophagy inhibition in U2OS cells treated with bated with CM from postslippage U2OS cells expressing shATG5 nocodazole or paclitaxel increased cell death postslippage showed a reduction of these cells capable of invasion compared (Fig. 4D) and reduced cell viability (Fig. 4E). Consistent with with control CM (Fig. 5C). To assess whether cytokines were this, clonogenic assays showed reduction of long-term cell sur- required for SASP-mediated paracrine signaling functions in vitro, vival following autophagic inhibition (Fig. 4F). Autophagy has we used CM from IL1b and IL8-deficient postslippage cells (Sup- been described to mediate degradation of lamin B1 (29). Inter- plementary Fig. S8C) for gene expression analysis of invasion estingly, nocodazole-treated RPE-1 cells pretreated with Baf A1 and migration-related markers. Depletion of either IL1b or IL8 prevented lamin B1 degradation at day 3 and day 6 (Supplemen- or both cytokines from the CM resulted in a significant decrease tary Fig. S6B and S6C). Notably, these differences were not due to in invasiveness compared with control CM (Supplementary changes at the transcriptional levels as our RT-PCR results exclud- Fig. S8D). In addition, decreased fibronectin expression was ed this possibility (Supplementary Fig. S6D). observed in all conditions (Supplementary Fig. S8E), whereas a To determine whether these findings could be extrapolated to decrease in MMP-9 was only observed upon double knockdown other antimitotic drugs, we assayed cells treated with nocodazole, of IL1b and IL8 (Supplementary Fig. S8E). Supplementation of paclitaxel, Mon and ZM for the apoptotic marker cleaved PARP IL1b or IL8 to postslippage CM collected under autophagy inhi- following ATG5 knockdown. Interestingly, the increase in cell bition conditions proved sufficient to rescue the reduction in death was restricted to MT-targeting drugs nocodazole and pac- invasiveness (Supplementary Fig. S8F). Taken together, this litaxel (Fig. 4G). In addition, autophagy inhibition increased suggests that autophagy-enabled production of IL1b and IL8 is DNA damage (as determined by gH2AX foci and 53BP1 protein crucial for postslippage senescent cells to promote paracrine levels) compared with control (Fig. 4G–I). Enhanced replication tumorigenic progression.

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To examine whether the in vitro migratory and invasive treatment. Molecular assessment of tumor sections derived from reduction following autophagic inhibition could be extrapo- mice xenografts showed more robust LC3 and p62 puncta accu- latedtometastaticinhibitionin vivo, we used the zebrafish mulation in the combination drug-treated mice compared with model of malignancy to monitor migration. The transparency nocodazole or paclitaxel treatment alone (Supplementary of the zebrafish embryo provides the unique ability to visualize Fig. S10B and S10C), indicating efficient autophagic inhibition. in vivo migratory changes of tumor cells in real time. U2OS In conclusion, we propose the model outlined in Fig. 6E. In H2B-GFP–expressing cells were incubated with CM from either response to the prolonged mitotic arrest induced by antimitotic cycling or postslippage cells expressing shATG5 or control for drugs, cells undergo either mitotic cell death or mitotic slippage. two days before injection into zebrafish embryos. H2B-GFP– In cells postslippage, autophagy is induced through ER stress- and expressing cells incubated with postslippage CM were observed UPR-mediated regulation of the AMPK/mTOR/ULK1 axis. This to migrate out from the injection site and metastasize further contributes to senescence and SASP production, which confers than those with control CM (Fig. 5D and E). This metastatic protumorigenic potential in a paracrine manner. Conversely, potential was significantly reduced following autophagic inhi- following autophagy inhibition, senescence is bypassed and bition via shATG5 (Fig. 5D and E). These observations further postslippage cells undergo death in a p53-dependent manner. support the role of autophagy-dependent SASP secretion in paracrine migration and invasion in vivo. Discussion Combination treatment of antimitotic drugs and autophagy The success of antimitotic therapies, often used as first-line inhibitor arrests tumor growth and is dependent on p53 status treatment of several malignancies (1), is limited due to acquired As p53 is a well-known regulator of cellular senescence (31), we resistance. The fate of cells following mitotic slippage, albeit hypothesized that p53 status could be an important predictor of representing a route of escape from mitotic arrest and mitotic response to combinatorial treatment of nocodazole or paclitaxel cell death, has not been extensively studied. Here, we show that with autophagy inhibition. Increased DNA damage (as measured multinucleated tetraploid cells that accumulate postslippage can by gH2AX protein levels) and a corresponding increase in cell undergo senescence and drive paracrine tumorigenic effects both death (by cell viability assays and cleaved PARP) were detected in in vitro and in vivo in an autophagy-dependent manner. U2OS cells postslippage with depleted p53 (shp53) compared Although prior seminal work on the autophagy-senescence with control (Supplementary Fig. S9A and S9B). In addition, we connection by Young and colleagues (32) showed that autopha- observed that HCT116 cells with wild-type (WT) p53 showed gic inhibition delayed the senescence phenotype following onco- increased cell death upon combination treatment compared with gene activation, our work demonstrates that inhibition of autop- p53-deficient cells (Fig. 6A). Cancer cells lines with dominant- hagy postslippage results in senescence bypass and accelerated cell negative p53 mutations, namely MDA-MB-231 breast cancer cells death. The most compelling evidence for senescence bypass after with p53 R280K, PanC1 pancreatic cancer cells with p53 R273H autophagy inhibition was increased entry into S-phase, the induc- and PaCa2 pancreatic cancer cells with p53 R248H also showed tion of DNA damage and replication stress, and reduction in cell less sensitivity to combination treatment compared with MCF7 viability in postslippage cells collaterally. breast carcinoma cells with WT p53 (Supplementary Fig. S10A). Autophagy has also been implicated as a potential inhibitory This suggests that cells with intact p53 might respond more regulator of senescence (33). How does one reconcile the fact that favorably to combinatorial treatment. autophagy can both activate and inhibit cellular senescence? Kang To test whether these findings could be extrapolated to an in vivo and colleagues (34) provide an explanation for these conflicting þ þ model, we treated mice xenografted with HCT116 cells (p53 / or results by describing the differential regulation of senescence via p53 / ) with combination treatment of either nocodazole or selective versus general autophagy. This is achieved by transcrip- paclitaxel and autophagic inhibitor chloroquine (CQ). Combi- tion factor GATA4, which the authors established to be a senes- nation treatment (nocodazole þ CQ or nocodazole þ CQ) cence regulator (34). GATA4 bound to autophagy adaptor p62 is compared with control nocodazole or paclitaxel alone signifi- degraded by selective autophagy under nonsenescent conditions. cantly decreased tumor growth and final tumor weight (Fig. 6B– This selective autophagy is suppressed upon senescence induction D), particularly in mice xenografts with WT p53, suggesting that where GATA4 is stabilized due to decreased GATA4–p62 inter- tumors with intact p53 were most sensitive to combination action. GATA4 stability triggers a series of events which ultimately

Figure 4. Autophagy mediates postslippage cell fate. A, Representative images of U2OS cells stably expressing shCtrl or shATG5 stained with SA-b-gal after 72-hour nocodazole (Noc) treatment. Plot shows percentage of SA-b-gal–positive cells. One-hundred cells per condition and data are mean SD of three independent experiments. Scale bar, 10 mm. B, U2OS cells were transfected with siCtrl or siATG5 followed by nocodazole treatment for 72 hours. Proliferation was assessed by the BrdU incorporation assay. Plot shows percentage of BrdU-positive multinucleated cells. One-hundred cells per condition and data are mean SD of three independent experiments. Scale bar, 20 mm. C, IB shows efﬁciency of ATG5 knockdown in U2OS cells transfected with siCtrl or siATG5 followed by nocodazole treatment for 72 hours. D, U2OS shCtrl or shATG5 cells carrying H2B-GFP were treated with nocodazole for 48 hours. Postslippage multinucleated cells were tracked individually by time-lapse microscopy for another 2 days. Red, cells that die postslippage. Black, cells that arrest postslippage. Fifty cells per condition and data are mean SD of two independent experiments. E, U2OS or RPE-1 cells were treated with nocodazole or paclitaxel in absence or presence of Baf A1 for 72 hours. Cell viability was assessed by CellTiter 96 One Solution Proliferation assay. F, Experimental scheme of clonogenic cell survival assay. Left, representative images of U2OS cells treated with the indicated drugs followed by crystal violet staining. Right, plot shows number of colonies counted using GelCount software. Eight wells per condition of 2 independent experiments. G, U2OS shCtrl or shATG5 cells were treated with indicated drugs for 72 hours and subjected to IB analysis. H, U2OS shCtrl or shATG5 cells were treated with nocodazole for 72 hours and subjected to IF. Plot shows percentage of gH2AX-positive multinucleated cells. One-hundred cells per condition and data are mean SD of two independent experiments. Scale bar, 20 mm. I, U2OS shCtrl or shATG5 cells were treated with nocodazole for 72 hours and subjected to IB analysis (, P < 0.05; , P < 0.005; , P < 0.001; , P < 0.0001; ns, not signiﬁcant by Student t test).

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Figure 5. Autophagy confers paracrine tumorigenic potential by regulating SASP. A, Luminex cytokine assay of supernatant obtained from U2OS cells treated with nocodazole in absence or presence of Baf A1 for 72 hours. B, Representative images of choroidal sprouts formed using indicated CM generated from U2OS shCtrl or shATG5 cells. Plot shows sprouting ratio (area of vascular sprouting/area of the explant). Minimum 6 explants analyzed per treatment. C, U2OS H2B-GFP cells were cultured with indicated CM from U2OS shCtrl or shATG5 for 48 hours. Invasive ability was assessed by transwell assay. Scale bar, 200 mm. Plot shows percentage of invasive cells. Five random fields were taken per condition and data are mean SD of three independent experiments. D, U2OS H2B-GFP cells were cultured with indicated CM generated from U2OS shCtrl or shATG5 cells, followed by injection into zebrafish embryo. All tumor foci observed in all tested larvae, belonging to each treatment group (presented as n on the graph), is presented as a scatter plot. Foci in one larva is represented by one color. "n" denotes number of injected embryos from three biological replicates. E, Quantitative comparison of metastatic ability upon indicated treatments was measured by invasive index (top) and migration index (bottom). , P < 0.05; , P < 0.005; , P < 0.001; , P < 0.0001 and ns, not significant by Student t test.

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Figure 6. Synergistic inhibition of tumor growth by antimitotic drugs and autophagy inhibitors depends on p53 status. A, HCT116 wt or p53/ cells were treated with the indicated drugs for 72 hours. Cell viability was assessed by CellTiter 96 One Solution Proliferation assay. Data are mean SD of three independent experiments. B, Representative images of xenograft tumors harvested from nude mice after two weeks of indicated treatment. Drug dosage used: autophagy inhibitor, Chloroquine (CQ): 30 mg/kg, nocodazole: 10 mg/kg, and paclitaxel (PTX): 10 mg/kg. C, Effect of combinatorial treatment on the growth of xenograft tumors generated by subcutaneous injection of HCT116 wt or p53/ cells in nude mice. Data are mean of SD (n ¼ 10 tumors per group). D, The weight of tumor after two weeks of indicated treatment. Dots represent individual tumor weight, n ¼ 10 tumors per group (, P < 0.05; , P < 0.005; , P < 0.001, ns, not signiﬁcant by Student t test). E, Proposed model of autophagy-mediated tumorigenesis upon antimitotic treatment. Upon treatment with antimitotic drugs, autophagy is induced via AMPK/mTOR/ULK1 axis activated by ER stress. Autophagy contributes to senescence and SASP production. This confers protumorigenic potential in a paracrine manner. Conversely, following autophagy inhibition senescence is bypassed and postslippage cells undergo death.

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leads to activation of NFkB for SASP production, thus facilitating thereby contributing to the systemic side effects of chemotherapy. senescence (34). In addition, autophagy has been described to Hence, several approaches targeting or bypassing senescence mediate degradation of lamin B1 (29). The loss of lamin B1, as during chemotherapy to reduce the likelihood of acquired resis- described before (12) and also shown in our study, is a senes- tance and chemotoxicity by redirecting cells toward apoptosis cence-associated marker. Inhibition of autophagy or LC3-lamin have been attempted (42). In addition, the conviction that senes- B1 interaction prevented oncogene-induced lamin B1 loss and cence is a truly irreversible process has been challenged as stem- attenuated senescence (29), and we observed similar block in like aggressive tumor-initiating cells have emerged from senescent lamin B1 degradation upon autophagy inhibition. It will be worth cells (43, 44). Interestingly, senescence revertants (without stem- exploring whether LC3–lamin B1 interaction and GATA4 con- like properties) have recently been shown to contain a subset of tribute to autophagy-induced senescence in our context. senescence-activated genes that can contribute to aggressiveness Our findings demonstrate that autophagy is induced during of the revertants (45). mitotic slippage through the PERK–AMPK axis of the ER stress Our results establish a key mechanism underlying cell fate response. Inhibition of ER stress decreased the rate of LC3-II after mitotic slippage and suggest a novel strategy combining conversion by preventing AMPK phosphorylation. Although cytotoxic drugs with autophagy inhibition to tackle undesired some studies indicate that ER stress prevents cellular senescence effects of mitotic slippage–induced senescence. It is conceivable by upregulating autophagy (35), there is compelling evidence to that senescent cells rely on autophagy as a nutrient source support the opposite. Using a mouse model of B-cell lymphoma to support cellular metabolism and survival, as well as for treated with the DNA-damaging agent cyclophosphamide, the autophagy-dependent clearance of targeted substrates. This Schmitt and colleagues' group (36) recently reported that senes- dependence could render them selectively vulnerable to autop- cent cells rely on autophagy to cope with SASP-coupled proteo- hagic inhibition. Our in vivo zebrafish and mice xenograft toxic stress. This proteotoxic stress can lead to transcriptional tumor models showed tumor growth arrest upon synergistic activation of UPR genes and autophagy induction (25), rendering autophagy inhibition and antimitotic drug treatment. The the senescent cells sensitive to autophagic inhibition. In addition, success of drug combination treatment was dependent on the autophagy might further be maintained as part of the innate status of p53, where tumors with wild-type p53 show greater immune response against cytoplasmic chromatin fragments asso- sensitivity to combinatorial treatment (46). This lends support ciated with senescence through the cGAS–STING pathway (37), as to the idea that p53 status is an important factor influencing cGAS directly interacts with Beclin-1 (38) to clear cytosolic DNA in therapeutic success and could be used as a potential biomarker an autophagy-dependent manner. In support of this, the cGAS for patient stratification, with exclusion of patients carrying and/or STING pathway has been reported by multiple groups to p53-mutant tumours. Currently, the autophagy inhibitors be involved in induction of senescence and/or SASP (37, 39). CQ and hydroxychloroquine (HCQ), clinically approved Postslippage SASP factors conferred paracrine protumorigenic for treatment of malaria, and additional newly discovered, phenotypic effects such as migration, invasion, and vasculariza- more potent autophagy-modulating compounds are showing tion on neighboring cells. Proliferation of cells incubated with promising results in clinical trials for the safe use to overcome CM from postslippage cells was unaffected. As it has been shown chemotherapeutic resistance (47, 48). that SASP composition and quantity is dependent on cell type Our studies also point to regimens targeting senescence and and mode of senescence induction (8), this suggests that post- SASP as potential combinatorial strategies with antimitotic drugs. slippage SASP may serve to play a "secondary" protumorigenic Although our current work focuses on MT-targeting drugs, we role in cells where migration, invasion, and angiogenesis are anticipate other classes of chemotherapeutic drugs to have similar uncoupled from cell proliferation thereby leading to the devel- roles in engendering non-cell–autonomous tumor progression. opment of a more malignant phenotype in cancer cells (40). It is Hence, it will be of interest to identify and delineate signaling possible that once neighboring cells become transformed and molecules and pathways leading to SASP and protumorigenic stimulated to proliferate, postslippage SASP then plays a role function postslippage. Future studies will focus on nodal points in to further enhance the tumorigenic capabilities of these cells, the "slippage-autophagy-senescence-SASP-tumor progression" demonstrating that mitotic slippage–induced senescence could axis as these could potentially serve as beneficial therapeutic serve as a conduit for malignant transformation and antimitotic targets in combination with antimitotic drug therapy. Interest- therapy resistance. ingly, induction of cellular senescence is often correlated with In contrast, multiple studies have reported the role of autop- polyploidy (49). It has been posited that polyploid tumors with hagy in promoting the elimination of tumor cells through mod- elevated genomic content tend to become resistant to chemo- ulation of the inflammatory response (41). The autophagy- therapy due to rapid tumor evolution (49). In addition, near- dependent inflammatory response is bidirectional and context- tetraploid cancer cells have recently been shown to exhibit dependent. To enhance efficacy of antimitotic therapies, one enhanced invasiveness (50). It will be of interest to determine might not only aim to eliminate protumorigenic SASP, but may whether high ploidy status per se is able to contribute to SASP- also provoke desirable immunogenic cell death and clearance induced tumor progression. by the induction of antitumorigenic SASP. Therefore, it will be important that future studies test the impact of mitotic slippage– Disclosure of Potential Conflicts of Interest induced SASP expression on the tumor microenvironment and No potential conflicts of interest were disclosed. host immune system in a spontaneous tumor model. In addition, targeting of senescent cells seems not only relevant Authors' Contributions for tumor cells and their microenvironment, but also for non- Conception and design: B. Cheng, K. Guo, X. Wang, J.E. Connolly, K.C. Crasta cancerous cells. The Campisi and colleagues' group (9) recently Development of methodology: A.X.F. Wong, B. Cheng, K. Guo, K.J. Lim, showed that cytotoxic drugs induce senescence in normal cells J.B.K. Khoo, B.T. Chua, I. Sinha, J.E. Connolly, K.C. Crasta

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Acquisition of data (provided animals, acquired and managed patients, Acknowledgments provided facilities, etc.): R. Jakhar, M.N.H. Luijten, A.X.F. Wong, This research is supported by the National Research Foundation, Prime B. Cheng, K. Guo, S.P. Neo, B. Au, M. Kulkarni, K.J. Lim, J. Maimaiti, Minister's Ofﬁce, Singapore, under its NRF Fellowship Programme (NRF Award H.C. Chong, E.H. Lim, T.B.K. Tan, K.W. Ong, Y. Sim, J.B.K. Khoo, I. Sinha, no. NRF-NRFF2013-10), Nanyang Assistant Professorship Grant, Nanyang J.E.Connolly,J.Gunaratne,K.C.Crasta Technological University and the Singapore Ministry of Education Academic Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Research Fund Tier 1 (Grant no.: 2015-T1-002-046-01; provided to K. Crasta). computational analysis): R. Jakhar, M.N.H. Luijten, A.X.F. Wong, B. Cheng, We are grateful to Sixun Chen and other members of the Crasta Lab for K. Guo, M. Kulkarni, K.J. Lim, H.C. Chong, B.T. Chua, I. Sinha, X. Wang, valuable comments and help with manuscript preparation. We thank AMPL for J.E. Connolly, J. Gunaratne, K.C. Crasta histology services, and G.L. Siok and X.L.R. Hai for technical support in MS analysis. Writing, review, and/or revision of the manuscript: R. Jakhar, M.N.H. Luijten, A.X.F. Wong, B. Cheng, K.J. Lim, I. Sinha, J.E. Connolly, J. Gunaratne, K.C. Crasta The costs of publication of this article were defrayed in part by the payment of Administrative, technical, or material support (i.e., reporting or organizing page charges. This article must therefore be hereby marked advertisement in data, constructing databases): A.X.F. Wong, B. Cheng, K. Guo, S.P. Neo, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. J. Maimaiti, J.T.S. Ho, J.E. Connolly, J. Gunaratne, K.C. Crasta Study supervision: J.E. Connolly, K.C. Crasta Other (contributed to mass spectrometry analysis): S.P. Neo, J. Gunaratne Received January 8, 2018; revised June 3, 2018; accepted July 10, 2018; Other (performed biopsies to obtain material): J.S.L. Wong published ﬁrst July 23, 2018.

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1640 Mol Cancer Res; 16(11) November 2018 Molecular Cancer Research

Autophagy Governs Protumorigenic Effects of Mitotic Slippage− induced Senescence

Rekha Jakhar, Monique N.H. Luijten, Alex X.F. Wong, et al.

Mol Cancer Res 2018;16:1625-1640. Published OnlineFirst July 23, 2018.

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