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• W2886911223 abstract "Research Article14 August 2018Open Access Source DataTransparent process CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer Murugan Kalimutho Corresponding Author Murugan Kalimutho [email protected] orcid.org/0000-0002-0772-8673 QIMR Berghofer Medical Research Institute, Herston, Qld, Australia School of Natural Sciences, Griffith University, Nathan, Qld, Australia Search for more papers by this author Debottam Sinha Debottam Sinha QIMR Berghofer Medical Research Institute, Herston, Qld, Australia School of Natural Sciences, Griffith University, Nathan, Qld, Australia Search for more papers by this author Jessie Jeffery Jessie Jeffery QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Katia Nones Katia Nones QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia Search for more papers by this author Sriganesh Srihari Sriganesh Srihari Computational Systems Biology Laboratory, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia Search for more papers by this author Winnie C Fernando Winnie C Fernando QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Pascal HG Duijf Pascal HG Duijf University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Claire Vennin Claire Vennin Cancer Division, Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Sydney, NSW, Australia Faculty of Medicine, St Vincent's Clinical School, University of NSW, Sydney, NSW, Australia Search for more papers by this author Prahlad Raninga Prahlad Raninga QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Devathri Nanayakkara Devathri Nanayakkara QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Deepak Mittal Deepak Mittal QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Jodi M Saunus Jodi M Saunus QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Centre for Clinical Research, The University of Queensland, Herston, Qld, Australia Search for more papers by this author Sunil R Lakhani Sunil R Lakhani Centre for Clinical Research, The University of Queensland, Herston, Qld, Australia School of Medicine, The University of Queensland, Herston, Qld, Australia Pathology Queensland, The Royal Brisbane and Women's Hospital, Herston, Qld, Australia Search for more papers by this author J Alejandro López J Alejandro López QIMR Berghofer Medical Research Institute, Herston, Qld, Australia School of Natural Sciences, Griffith University, Nathan, Qld, Australia Search for more papers by this author Kevin J Spring Kevin J Spring Liverpool Clinical School, University of Western Sydney, Liverpool, NSW, Australia Ingham Institute, Liverpool Hospital, Liverpool, NSW, Australia South Western Sydney Clinical School, University of New South Wales, Liverpool, NSW, Australia Search for more papers by this author Paul Timpson Paul Timpson Cancer Division, Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Sydney, NSW, Australia Faculty of Medicine, St Vincent's Clinical School, University of NSW, Sydney, NSW, Australia Search for more papers by this author Brian Gabrielli Brian Gabrielli University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia Mater Research Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Nicola Waddell Nicola Waddell QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Kum Kum Khanna Corresponding Author Kum Kum Khanna [email protected] orcid.org/0000-0001-8650-5381 QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Murugan Kalimutho Corresponding Author Murugan Kalimutho [email protected] orcid.org/0000-0002-0772-8673 QIMR Berghofer Medical Research Institute, Herston, Qld, Australia School of Natural Sciences, Griffith University, Nathan, Qld, Australia Search for more papers by this author Debottam Sinha Debottam Sinha QIMR Berghofer Medical Research Institute, Herston, Qld, Australia School of Natural Sciences, Griffith University, Nathan, Qld, Australia Search for more papers by this author Jessie Jeffery Jessie Jeffery QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Katia Nones Katia Nones QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia Search for more papers by this author Sriganesh Srihari Sriganesh Srihari Computational Systems Biology Laboratory, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia Search for more papers by this author Winnie C Fernando Winnie C Fernando QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Pascal HG Duijf Pascal HG Duijf University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Claire Vennin Claire Vennin Cancer Division, Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Sydney, NSW, Australia Faculty of Medicine, St Vincent's Clinical School, University of NSW, Sydney, NSW, Australia Search for more papers by this author Prahlad Raninga Prahlad Raninga QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Devathri Nanayakkara Devathri Nanayakkara QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Deepak Mittal Deepak Mittal QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Jodi M Saunus Jodi M Saunus QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Centre for Clinical Research, The University of Queensland, Herston, Qld, Australia Search for more papers by this author Sunil R Lakhani Sunil R Lakhani Centre for Clinical Research, The University of Queensland, Herston, Qld, Australia School of Medicine, The University of Queensland, Herston, Qld, Australia Pathology Queensland, The Royal Brisbane and Women's Hospital, Herston, Qld, Australia Search for more papers by this author J Alejandro López J Alejandro López QIMR Berghofer Medical Research Institute, Herston, Qld, Australia School of Natural Sciences, Griffith University, Nathan, Qld, Australia Search for more papers by this author Kevin J Spring Kevin J Spring Liverpool Clinical School, University of Western Sydney, Liverpool, NSW, Australia Ingham Institute, Liverpool Hospital, Liverpool, NSW, Australia South Western Sydney Clinical School, University of New South Wales, Liverpool, NSW, Australia Search for more papers by this author Paul Timpson Paul Timpson Cancer Division, Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Sydney, NSW, Australia Faculty of Medicine, St Vincent's Clinical School, University of NSW, Sydney, NSW, Australia Search for more papers by this author Brian Gabrielli Brian Gabrielli University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia Mater Research Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Nicola Waddell Nicola Waddell QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Kum Kum Khanna Corresponding Author Kum Kum Khanna [email protected] orcid.org/0000-0001-8650-5381 QIMR Berghofer Medical Research Institute, Herston, Qld, Australia Search for more papers by this author Author Information Murugan Kalimutho *,1,2,‡, Debottam Sinha1,2,‡, Jessie Jeffery1, Katia Nones1,3, Sriganesh Srihari4, Winnie C Fernando1, Pascal HG Duijf5, Claire Vennin6,7, Prahlad Raninga1, Devathri Nanayakkara1, Deepak Mittal1, Jodi M Saunus1,8, Sunil R Lakhani8,9,10, J Alejandro López1,2, Kevin J Spring11,12,13, Paul Timpson6,7, Brian Gabrielli5,14, Nicola Waddell1 and Kum Kum Khanna *,1 1QIMR Berghofer Medical Research Institute, Herston, Qld, Australia 2School of Natural Sciences, Griffith University, Nathan, Qld, Australia 3Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia 4Computational Systems Biology Laboratory, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Qld, Australia 5University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia 6Cancer Division, Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Sydney, NSW, Australia 7Faculty of Medicine, St Vincent's Clinical School, University of NSW, Sydney, NSW, Australia 8Centre for Clinical Research, The University of Queensland, Herston, Qld, Australia 9School of Medicine, The University of Queensland, Herston, Qld, Australia 10Pathology Queensland, The Royal Brisbane and Women's Hospital, Herston, Qld, Australia 11Liverpool Clinical School, University of Western Sydney, Liverpool, NSW, Australia 12Ingham Institute, Liverpool Hospital, Liverpool, NSW, Australia 13South Western Sydney Clinical School, University of New South Wales, Liverpool, NSW, Australia 14Mater Research Institute, Translational Research Institute, The University of Queensland, Brisbane, Qld, Australia ‡These authors contributed equally to this work *Corresponding author. Tel: +61 38453772; E-mail: [email protected] *Corresponding author. Tel: +61 7 3362 0338; E-mail: [email protected] EMBO Mol Med (2018)10:e8566https://doi.org/10.15252/emmm.201708566 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in in vivo models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers. Synopsis By integrating analyses of multiple in vitro and in vivo breast cancer models, this study provides direct evidence for CEP55-mediated protection of aneuploidy. Targeting MEK1/2-PLK1 axis in CEP55-MYC-dependent basal-like, triple-negative breast cancers could be a novel treatment strategy. CEP55 overexpression is associated with poor clinical outcomes in breast cancer. CEP55 dictates cell fate during perturbed mitosis. High levels of CEP55 are protective in aneuploid cells. Loss of CEP55 primes premature CDK1/Cyclin B activation and apoptosis upon treatment with anti-mitotic drugs. Cep55 is a marker for rationale use of combined MEK1/2-PLK1 inhibition in triple-negative breast cancers. Introduction Centrosomal proteins have long been recognized as scaffold proteins, regulating both the mitotic spindle and microtubule organization, and hence are critical for cell cycle progression, reviewed in Kumar et al (2013). CEP55 (also known as FLJ10540/C10orf3) is a coiled-coil centrosomal protein originally identified as an indispensable regulator of cytokinesis, the final stage of cell division that results in the physical separation of two daughter cells (Fabbro et al, 2005; Martinez-Garay et al, 2006; Zhao et al, 2006; van der Horst & Khanna, 2009; van der Horst et al, 2009). CEP55 localizes to the centrosome throughout the cell cycle, to the mitotic spindle during mitosis and the midbody during cytokinesis (Lee et al, 2008). Cytokinesis is a tightly controlled process during cell division requiring multicomponent subunits that are recruited to the midbody in a CEP55-dependent manner (Fabbro et al, 2005; Bastos & Barr, 2010; Mondal et al, 2012; Agromayor & Martin-Serrano, 2013). These events are primarily mediated by the Endosomal Sorting Complex Required for Transport (ESCRT) machinery to ensure equal segregation of cytoplasmic contents between daughter cells (Lee et al, 2008). Failure to coordinate these events results in primary genetic lesions that promote genomic instability and tumorigenesis (Agromayor & Martin-Serrano, 2013). CEP55 is expressed at low levels in most normal human tissues except testis, and its dysregulation is linked to multiple disease states (Sakai et al, 2006; Chen et al, 2009b; Inoda et al, 2009; Waseem et al, 2010; Janus et al, 2011; Shiraishi et al, 2011; Chen et al, 2012; Hwang et al, 2013; Jeffery et al, 2015; Zhang et al, 2016a; Bondeson et al, 2017; Frosk et al, 2017). It is associated with aggressive behavior in in vivo models, is an independent marker of poor clinical outcome in various malignancies, and has been recognized as a strong candidate for vaccine development in breast and colorectal cancers (Inoda et al, 2009, 2011a,b). In terms of its mechanistic involvement in cancers, CEP55 increases anchorage-independent growth, migration, and invasion in vitro and promotes tumor formation in nude mice, possibly through VEGFA-PI3K/AKT signaling (Chen et al, 2007; Inoda et al, 2009; Hwang et al, 2013). Gene expression studies implicated CEP55 in progression from in situ to invasive breast cancer (Ma et al, 2003), and overexpression in primary breast tumors is a marker of chromosomal instability and poor prognosis (Carter et al, 2006; Fournier et al, 2006). Therefore, it appears that CEP55 overexpression plays a pivotal role in tumorigenesis, likely through the emergence of aneuploidy. However, the mechanism of how CEP55 mediates genomic instability, aneuploidy, and tumorigenesis has remained elusive. In this study, we provide the first experimental evidence directly linking CEP55-dependent aneuploidy to breast cancer survival. Using large breast datasets with clinical follow-up information, we confirmed that high levels of CEP55 mRNA associate with poor clinical outcomes. Knockdown of CEP55 in breast cancer cells in vitro significantly reduced the number of aneuploid cells, induced cell death during perturbed mitosis, and sensitized cells to anti-mitotic agents. Rapid onset of G2/M entry due to premature CDK1/cyclin B activation primed cell death following treatment with anti-mitotic agents in a CEP55-dependent manner. Furthermore, we found that CEP55 is a downstream effector of mitogen-activated protein kinase (MAPK)-MYC signaling. Dual inhibition of MAPK signaling (MEK1/2 inhibition) and the mitotic pathway (PLK1 inhibition) synergistically reduced the outgrowth of both murine and human breast cancer cells. These results provide a rationale for clinically targeting CEP55-dependent pathways in basal-like, triple-negative breast tumors for better treatment efficacy. Results CEP55 overexpression is associated with poor outcome in breast cancer Although CEP55 is ubiquitously overexpressed in many human cancers (Jeffery et al, 2015), a detailed molecular understanding of its role in tumorigenesis has remained elusive. We analyzed CEP55 expression using the publically available Gene expression-based Outcome for Breast cancer Online (GOBO) database (n = 1,881; Ringner et al, 2011). We found that CEP55 mRNA expression is associated with the PAM50 breast cancer molecular subtypes (Luminal A, Luminal B, HER2, and basal-like), with the basal-like subtype exhibiting significantly higher expression of CEP55 compared to other subtypes (P < 0.0001; Fig EV1A, available online). This increased expression of CEP55 was also associated with high-grade tumors (P < 0.0001; Fig EV1B) and estrogen receptor (ER) status (P < 0.0001; Fig EV1C). CEP55 high expression was significantly associated with poor overall survival (P = 0.00102), relapse-free survival (P < 0.00001), and distant metastasis-free survival (P = 0.01135) (Fig EV1D–F). Similarly, a strong association between high CEP55 expressing tumors and poor survival was observed when we used a larger patient dataset from KMPlotter (n = 4,142; Appendix Fig S1A; Gyorffy et al, 2010). CEP55 is part of a proliferation/mitotic gene signature suggesting that the observed differences in patient survival could be due to its association with proliferation. To rule out this possibility, we normalized the expression value of CEP55 with key proliferation markers, KI67 and PCNA using the TCGA (The Cancer Genome Atlas) dataset (n = 492) (Cancer Genome Atlas, 2012). This confirmed that CEP55 expression was significantly higher in breast cancer patients compared to normal breast tissue independent of proliferation (P < 0.0001; Appendix Fig S1B and C). Collectively, these data provide compelling evidence that high expression of CEP55 mRNA is associated with poor clinical outcomes in breast cancer and therefore could be a novel target for therapeutic intervention. Click here to expand this figure. Figure EV1. Clinical correlation of CEP55 mRNA expression in breast cancer datasets A–C. Relationship between CEP55 mRNA expression (Log 2 expression) and (A) breast cancer intrinsic molecular subtypes, (B) histological grade, and (C) estrogen receptor (ER) status evaluated through the GOBO online tool (http://co.bmc.lu.se/gobo/; Ringner et al, 2011). Number of patients used for the analysis was indicated at the bottom of each panel. D–F. Top panel, association of CEP55 expression with clinical outcome for overall survival (D), relapse-free survival (E) and distant metastasis-free survival (F) determined using the GOBO datasets; bottom panel, corresponding multivariate parameters analyses. Patients were divided into CEP55 low and high expression. Download figure Download PowerPoint Differential expression of CEP55 regulates breast cancer cell proliferation and survival To help select suitable models for functional work, we first analyzed CEP55 expression in a published breast cancer cell line gene expression array dataset (n = 51 lines; Neve et al, 2006). Similar to clinical samples, CEP55 mRNA expression was higher in basal-like, triple-negative cell lines, particularly those with mesenchymal and invasive phenotypes (Appendix Fig S2A–C). Immunoblotting analysis showed a similar trend toward higher protein expression in basal-like lines (Fig 1A), but most striking was the higher expression observed in HRAS-transformed “AT” and “CA” derivatives of MCF10A compared to the near-normal (diploid) MCF10A parental cells (Fig 1B; Soule et al, 1990). To better understand the potential role of CEP55 in breast cancer, we transiently knockdown CEP55 with pooled siRNAs in a panel of breast cancer lines and noticed significantly reduced viability of 6/8 basal and 4/9 luminal/HER2 cell lines with cutoff of 50% inhibition, irrespective of their baseline CEP55 expression (Figs 1C and EV2A). Moreover, knockdown of CEP55 in two representative basal-like lines resulted in significant induction of cell death as evident by increased proportion of cells with sub-G1 DNA content (Fig EV2B). Figure 1. CEP55 regulates human breast cancer cell survival A, B. Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines (n = 25) and in progression series of MCF10A lines (MCF10AT and MCF10CA), respectively. Tubulin and COX-IV served as loading controls. C. A panel of selected breast cancer and near-normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean ± SEM of three independent experiments. D. Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA-MB-231 cells. Two isogenic lines (sh#2 and sh#8) were obtained using two different shRNA sequences as described in method section. COX-IV served as loading control. E. Effect of CEP55 knockdown on cell proliferation in MDA-MB-231 cells assessed using the IncuCyte ZOOM® live-cell imager (phase-only processing module). The percentage of cell confluence was determined using an IncuCyte mask analyzer. Graph represents the mean ± SEM of three independent experiments. F. Representative images of colony-forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA-MB-231 cells. G. Six-week-old female NOD/SCID cohorts of mice were injected in the 4th inguinal mammary fat pad with the control and CEP55 knockdown cells. Growth rate (area, mm2) of the tumors was measured using digital calliper. Differences in growth were determined using Student's t-test, ****P ≤ 0.0001. Graph represents the mean tumor area ± SEM, n = 6 mice/group. H. Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag-CEP55 expression construct and two isogenic lines were obtained (CEP55-#16 and CEP55-#17) with its respective empty vector (EV) transfected parental cells. COX-IV served as a loading control. I. Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM® live-cell imager as described in panel (E). Graph represents the mean ± SEM of two independent experiments. J. Representative images of colony-forming capacity at 14 days determined using crystal violet staining in empty vector (EV) and CEP55-overexpressing (CEP55#17 and CEP55#18) lines. K. Representative images of single-plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: cytokeratin 19; green: F-actin stained by Phalloidin and blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CEP55 regulates human breast cancer cell survival (related to Fig 1) A. Immunoblot analysis of CEP55 expression showing knockdown efficiency following 5 nM of pool siRNAs transfection at 72 h in representative triple-negative breast cancer lines. COX-IV as a loading control. B. Effect of CEP55 knockdown using siCEP55 (5 nM) on cell proliferation and death in both BT549 and MDA-MB-436, triple-negative cell lines. Proliferation was assessed using the IncuCyte ZOOM® live-cell imager. The percentage of cell confluence was determined using an IncuCyte mask analyzer (left panel), and apoptotic fractions (sub-G1 population) was determined by propidium iodide staining using flow cytometry and ModFit LT 4.0 software analysis (right panel). Graphs represent the mean ± SEM of two independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. C. Left, representative cytogram analysis of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles at day 30. Cells were exposed to doxycycline (2 μg/ml) twice weekly for 4 weeks. Middle, cell cycle distribution and right, sub-G1 analyses of corresponding data are shown. Graphs represent representative data from two independent experiments. *P ≤ 0.05. Mean ± SEM. D. Sequencing chromatograms of both wild-type and shRNA-resistant constructs verified by Sanger sequencing are shown. E. Top, immunoblot analysis of CEP55 expression in CEP55 knockdown MDA-MB-231 cells. The shRNA-resistant construct was transiently transfected with 1 μg of DNA in sh#8 cells, and CEP55 expression was determined after 48 h post-transfection. COX-IV served as loading control. Bottom, effect of CEP55 reintroduction on cell proliferation in CEP55-knockdown sh#8 cells assessed using the IncuCyte ZOOM® live-cell imager as described in panel (B). Graph represents the mean ± SEM of three independent experiments. F. Quantification of crystal violet intensity staining (absorbance value at 540 nM) on colony-forming capacity in both control and CEP55 knockdown in MDA-MB-231 cells. For colony formation assay, 1,000 cells were seeded. ****P ≤ 0.0001. Mean ± SEM. Download figure Download PowerPoint To further evaluate the impact of CEP55 on cellular proliferation and survival, we knockdown CEP55 in MDA-MB-231 cells (shows highest protein level in the panel of cell lines (Fig 1A) and exhibits invasive behavior in vitro/vivo) by stable transduction with doxycycline-inducible shRNAs that target different regions in the CEP55 transcript (Appendix Fig S2D). The inducible knockdown system used in this study was “leaky”, as evidenced by reduced CEP55 expression in both sh#2 and sh#8 polyclonal lines in the absence of doxycycline compared to scrambled shRNA-transfected cells (hereafter referred to as control) (Fig 1D and Appendix Fig S2D). Previously, we have shown that complete knockdown of CEP55 using siRNAs in Hela cells resulted in cytokinesis failure leading to multinucleation (Fabbro et al, 2005). However, partially knockdown of CEP55 in breast cancer cells using shRNAs (sh#2 or sh#8) showed no significant changes in the number of cells displaying cytokinesis failure although complete knockdown of CEP55 by growing cells continuously in presence of doxycycline over 30 days showed increased proportion of cells in G2/M phase with multinucleation concomitant with increased cell death (Fig EV2C), suggesting that multinucleated cells are not viable over a long period of time. Therefore, stable CEP55 (sh#2 and sh#8) knockdown pools with reduced CEP55 expression (without doxycycline induction) were used in further studies to minimize the pronounced negative impact of inducible-complete CEP55 knockdown on cytokinesis failure and loss of viability. Both lines with reduced CEP55 exhibited significantly delayed proliferation which could be rescued by transiently expressed shRNA-resistant CEP55 expression construct (P < 0.0001; Figs 1E and EV2D and E). We also found that anchorage-independent colony formation was reduced upon CEP55 knockdown (P < 0.0001; Figs 1F and EV2F). Since CEP55 stimulates cell migration and invasion in oral and lung cancers (Chen et al, 2009a), we assayed in vitro migration and invasion in the MDA-MB-231 derivatives and observed a significant reduction compared to control cells (P < 0.001; Appendix Fig S3A and B). Engrafting a partial CEP55-knockdown MDA-MB-231 derivative (sh#2) showed a significant reduction in tumor growth while a near-complete CEP55 reduction (sh#8) abrogates tumor formation in NOD/SCID mice (Fig 1G and Appendix Fig S3C; 3 mice/group are shown as an example), suggesting that CEP55 is essential for tumor formation. To complement these knockdown experiments, we studied the effect of Flag-CEP55 overexpression in MCF10A cells, a near-normal mammary epithelial line, and generated two isogenic lines expressing high levels of CEP55 (Fig 1H, CEP55—#16 and—#17) (level of ectopic CEP55 is comparable to endogenous CEP55 found in highly aggressive MDA-MB-231 cells). Initial characterization of these cells demonstrated a significant increase in proliferation (P < 0.01; Fig 1I) and migration (P < 0.0006; Appendix Fig S3D). Next, we tested whether the CEP55-overexpressing MCF10A cells had acquired an oncogenic phenotype by measuring their ability to form colonies in non-adherent conditions and form acinus-like structures on Matrigel. Both CEP55-overexpressing lines formed colonies more efficiently (P < 0.0001; Fig 1J, Appendix Fig S3E) and formed larger acinus-like spheroids on Matrigel compared to the empty vector (EV) transfected MCF10A cells (Fig 1K), and the latter are known to form growth-arrested structures with a well-defined lumen in the center (Pal & Kleer, 2014). Moreover, we found that CEP55-overexpressing lines exhibited decreased staining of cytokeratin 19, a main cytoskeleton protein of epithelial cells and failed to form a hollow lumen as compared to control (EV) transfected MCF10A cells, suggesting that CEP55 overexpression is sufficient to confer malignant phenotype to near-normal MCF10A cells. Collectively, these data suggest that CEP55 overexpression confers anchorage-independent growth and a survival advantage to breast cancer cells. High levels of CEP55 are protective in aneuploid cells CEP55 is part of the 70-gene chromosomal instability (CIN) signature that was associated with aneuploidy in several human cancers (Carter et al, 2006); thus, we questioned whether high CEP55 expression could confer tolerance to or promote aneuploidy (causal effect). The first scenario would result in reduction of aneuploidy in CEP55-knockdown cells, and in the second scenario, CEP55 may act as" @default.
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• W2886911223 title "<scp>CEP</scp> 55 is a determinant of cell fate during perturbed mitosis in breast cancer" @default.
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