Neuroepigenetics 7 (2016) 6–18

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Neuroepigenetics

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Gene knockdown of CENPA reduces sphere forming ability and stemness of glioblastoma initiating cells

Jinan Behnan a,1, Zanina Grieg b,c,1, Mrinal Joel b,c, Ingunn Ramsness c, Biljana Stangeland a,b,⁎ a Department of Molecular Medicine, Institute of Basic Medical Sciences, The Medical Faculty, University of Oslo, Oslo, Norway b Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway c Vilhelm Magnus Laboratory for Neurosurgical Research, Institute for Surgical Research and Department of Neurosurgery, Oslo University Hospital, Oslo, Norway article info abstract

Article history: CENPA is a -associated variant of H3 implicated in numerous malignancies. However, the Received 20 May 2016 role of this in glioblastoma (GBM) has not been demonstrated. GBM is one of the most aggressive Received in revised form 23 July 2016 . GBM initiating cells (GICs), contained within these tumors are deemed to convey Accepted 2 August 2016 characteristics such as invasiveness and resistance to therapy. Therefore, there is a strong rationale for targeting these cells. We investigated the expression of CENPA and other centromeric (CENPs) in Keywords: fi CENPA GICs, GBM and variety of other cell types and tissues. Bioinformatics analysis identi ed the signature: fi Centromeric proteins high_CENP(AEFNM)/low_CENP(BCTQ) whose expression correlated with signi cantly worse GBM patient Glioblastoma survival. GBM Knockdown of CENPA reduced sphere forming ability, proliferation and cell viability of GICs. We also tumor detected significant reduction in the expression of stemness marker SOX2 and the proliferation marker Glioblastoma initiating cells and therapeutic Ki67. These results indicate that CENPA might represent a promising therapeutic target for GBM treatment. targeting © 2016 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction (Sandberg et al., 2013). This analysis also revealed a 30-gene signature highly up-regulated in GICs that had an impact on survival of glioma Glioblastoma (GBM) is one of the most malignant human cancers patients (Sandberg et al., 2013). Recently we identified a with dismal prognosis. Despite multimodal therapy and inclusion of glioblastoma-specific coexpression module that consisted of PBK/TOPK, temozolomide, median survival remains unchanged (Helseth et al., CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/ 2010; Ronning et al., 2012). Glioblastoma initiating cells (GICs), a cell RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, population with stem cell properties (also referred to as glioblastoma MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A (Stangeland et al., stem cells, GSCs) harbored with within these tumors are deemed 2015). All nine in the module were highly up-regulated in GBM important for retaining GBM properties (Galli et al., 2004; Singh et al., and were predominantly involved in , cell-cycle and 2003; Vescovi et al., 2006). GICs are invasive (Cheng et al., 2011; Molina proliferation (Stangeland et al., 2015). The cumulative increased et al., 2010), promote tumor angiogenesis (Folkins et al., 2009; Das and expression of this nine-gene module correlated negatively with survival Marsden, 2013; Bao et al., 2006) and are resistant to irradiation and of GBM patients thus suggesting that they might represent potential chemotherapy (Bao et al., 2006; Chen et al., 2012; Liu et al., 2006). therapeutic targets (Stangeland et al., 2015). Two of the genes KIF15 and Consequently, there is a strong rationale for targeting these cells. KIF18A were involved in mitotic kinesis (Stangeland et al., 2015). KIF15 is We have compared in GICs to that in neural stem required for spindle function and has recently been targeted by using cells (NSCs) from the adult human brain, and identified genes and quinazolinedione and phthalimide (Mcdonald et al., 2004). KIF18A is biochemical pathways typically up-regulated in GICs (Sandberg et al., involved in congression and a specific inhibitor BTB1 that 2013; Stangeland et al., 2015). Although these two cell types share targetsthisgenewasrecentlydiscovered(Catarinella et al., 2009). common stem- and lineage-related markers, GICs show a more Knockdown of a further three genes: HMMR (Tilghman et al., 2014), heterogeneous gene expression and a number of pathways such as EZH2 (Suva et al., 2009)andPBK (Joel et al., 2015) resulted in severe Wnt signaling, and p53 signaling are dysregulated in GICs reduction of tumorigenic features of GICs. One of the nine genes, encoding a centromeric protein ‘A’ (CENPA), was highly up-regulated in GICs both at RNA and protein levels ⁎ Corresponding author. E-mail address: [email protected] (B. Stangeland). (Stangeland et al., 2015). CENPA, is a variant of (Sullivan et 1 These authors contributed equally and are listed alphabetically. al., 1994) that was firstly identified in scleroderma patients (Moroi et http://dx.doi.org/10.1016/j.nepig.2016.08.002 2214-7845/© 2016 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 7 al., 1980; Earnshaw and Rothfield, 1985). CENPA homologs have been bFGF twice a week. When the spheres reached approximately 100 μmin identified in organisms from yeast to human, where they serve as diameter they were dissociated into single cells. markers of centromeric identity (Verdaasdonk and Bloom, Tissue specimens from the adult human brain were dissociated 2011). During cell division the centromere recruits the that into single cells and cultured according to our FAILSAFE protocol (1% additionally attaches to the spindle. CENPA secures proper kinetochore FBS, 10 ng/ml bFGF and 20 ng/ml TGF α) that ensures robust function (Howman, 2000; Blower and Karpen, 2001; Oegema et al., long-term propagation of multipotent stem cells from the adult 2001) by recruiting the inner kinetochore proteins CENPC, CENPN and human brain (Murrell et al., 2013). CENPB (Guse et al., 2011; Carroll et al., 2009; Ando et al., 2002). The kinetochore interface consists of a large number of other centromere 2.2. Microarray analysis and public database mining proteins (CENPs) that form constitutive a centromere-associated network (CCAN) (for overview see McKinley and Cheeseman and the Hierarchical clustering with distance matrix and the Principal references therein) (McKinley and Cheeseman, 2016). CENPA is also Component Analysis were performed using J-Express software (Molmine, involved in response to DNA damage and ensures integrity Bergen,Norway).Fortheseanalyses we used our microarray expression (Mathew et al., 2014; Lawrence et al., 2015). Several recent reports data derived from GIC, neural fetal cells (NFCs), brain tissue and NSC implicate CENPA in numerous human cancers such as breast, liver, colon, cultures with submission numbers GSE60705, GSE53800, GSE41470 lung, blood, ovarian and pancreatic (Zhang et al., 2014; Habel et al., (encompassing GSE41467, GSE41390, and GSE41394), as well as the 2013; Toh et al., 2011; Rajput et al., 2011; Hu et al., 2010; Biermann et al., following publically available (GEO repository) microarrays encompass- 2007a; Biermann et al., 2007b; Furukawa et al., 2006; Li et al., 2007; ing: additional sets of GICs (GSE36426), astrocytes (GSE47515), breast Tomonaga et al., 2003; Qiu et al., 2013). Increased expression of CENPA stem cells and cell lines (GSE34987, GSE36102), iPS (GSE43364, gene correlates negatively with patient survival (Zhang et al., 2014; GSE43903, GSE41565), GBM cell lines (GSE43452), iPS derived partially Habel et al., 2013; Qiu et al., 2013). CENPA is also associated with the differentiated neurons (GSE42265), NSCs (GSE32658), ESCs and fibro- transformation of lymphoblasts by Epstein–Barr virus (Dai et al., 2012). blasts (GSE37077) and leukocytes (GSE42133). Although the significance of CENPA in human cancers has been For Fig. 2: The expression of eleven CENPs was analyzed in the documented its relevance in GBM and GICs remains to be demonstrated. previously defined subset of 200 GBM samples from TCGA (Verhaak et In this paper we investigated the expression of CENPA and several other al., 2010). PCA, HCA and HCA with distance matrix were performed CENPs in GBM, brain tissues, GICs, NSCs, a neural fetal cells (NFCs) line, using J-express, Molmine, Bergen, Norway. induced pluripotent stem cells (iPS), dopaminergic neurons and many Fig. 1D and Supplemental Fig. 2: For PCA analysis we downloaded other cell and tissue types using bioinformatics analysis. We also the following subsets from GEO superseries GSE27869: GSM688128 investigated the effect of CENPA gene knock-down on sphere forming (CENPA KD), GSM688129 (CENPB KD), GSM688130 (CENPC1 KD), ability, stemness and proliferation of GICs. GSM688131 (CENPE KD), GSM688132 (CENPF KD), GSM688133 (CENPH KD). The above-mentioned microarrays were from GE 2. Materials and methods Healthcare microarray platform GPL4044 (spotted oligonucleotide, CodeLink UniSet Human 20 K I Bioarray). Each culture featured only a 2.1. Tissue specimens and cell culture single CENP gene knockdown as specified. Bioinformatic analysis was performed in J-Express. The study was approved by the Regional committee for medical For calculation of statistical parameters, we used Graphpad Prism and health research ethics (REC South-East S-07321d). Biological (www.graphpad.com). material and personal health data have been collected and used in accordance with informed consent from patients included in the study 2.3. Western blot and quantification of protein expression and in compliance with the institutional guidelines pertaining to use of such issued by the department of research support at Oslo The cells were homogenized by triturating in Cell Extraction Buffer University Hospital. Tissue specimens were harvested from consent- (Mammalian cell extraction kit, Biovision, Milpitas, CA, USA) and ing patients and were obtained as a part of surgical procedures for centrifuged through a QIAshredder (Qiagen, Venlo, Netherlands). 20– treating GBM. Normal brain tissue was obtained from fresh human 40 μg of whole protein extract were mixed with loading buffer temporal lobes surgically resected to treat medically refractory (NuPAGE, Invitrogen) and loaded onto a 4–20% gradient Nu-PAGE gel epilepsy. All biopsy specimens were evaluated by neuropathologists. (Invitrogen). Protein gels were blotted onto 0.45 μm PVDF mem- Tumor biopsies underwent mechanical dissociation and branes (Invitrogen) and western blots were performed as previously Trypsin-EDTA (Gibco, Life Technologies, NYC, NY, USA) was added described (Stangeland et al., 2015). For western blot we used for enzymatic dissociation. Subsequently, 2 mg/ml human albumin following primary antibodies: Anti-CENPA (#2186, rabbit, 1:1000), (Octapharma pharmazeutika produktionges, Vienna, Austria) was Anti-ACTIN-B (#4967, rabbit, 1:1000) both from used to block the Trypsin effect and the cells were washed in L-15 technologies (CST). The secondary antibody was ECL Anti-rabbit (Lonza, Basel, Switzerland) before being plated in serum-free neuro- IgG-HRP (NA934, 1:10000, Amersham). Protein quantification was sphere medium containing 10 ng/ml basic fibroblast growth factor performed as previously described (Stangeland et al., 2015). Briefly, (bFGF) and 20 ng/ml epidermal growth factor (EGF) (both R&D Inc., CENPA and ACTB (actin) protein bands from the same lane were Minneapolis, MN, USA), B27-supplement (1:50, Invitrogen, Carlsbad, quantified using densitometry function in Photoshop. ACTB was used CA, USA), 100 U/ml Penicillin/streptomycin (Lonza), 1 ng/ml Heparin as a control for normalization. The Relative Protein Expression (RPE) (Leo Pharma, Ballerup, Denmark) and 8 mM Hepes (Lonza) in Dulbecco's values were calculated by normalizing CENPA band intensities to modified essential medium with nutrient mix F-12 and Glutamax those of ACTB. The RPE values for KDs were then normalized to (DMEM/F12, Invitrogen). Dissociated GBM biopsies grown as free- non-silencing control and used for comparison. floating spheres in serum free-medium containing mitogens (EGF and bFGF) are highly enriched for GICs. GIC culture T65 was previously 2.4. RNAi-mediated gene knockdown extensively characterized for stemness (CD133, CD44, SSEA-1/CD15, CXCR4, CD9, CD166, A2B5) and tumor forming capability by transplant- GIC culture T65 was used to establish stable CENPA knockdown ing into the brain of SCID mice (Stangeland et al., 2015; Joel et al., 2015). cultures KD1 (Clone ID V3LHS_403419), KD2 (V2LHS_150535), KD3 The cells were cultured in 75 cm2 non-treated flasks (Nunc, Roskilde, (V3LHS_313522), KD4 (V3LHS_403420) and a non-silencing (NS) Denmark) at a density of 10⁵ cells/ml and supplemented with EGF and shRNA as a control (RHS4346) (all from Open Biosystems, Thermo 8

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Scientific, Huntsville, AL, USA). Production of virus and transduction Supplemental Fig. 1). The expression of a variety of CENPs was were performed as previously described (Joel et al., 2015). The cells were analyzed using hierarchical cluster analysis (Fig. 1). In addition to selected on 1–3 μg/ml Puromycin (Sigma-Aldrich, St. Louis, MO, USA). GICs, NSC, GBM, commercial GBM cell lines and brain tissues we also included the following: neural fetal cells (NFCs), astrocytes, iPS, 2.5. Cell viability and sphere forming assay embryonic stem cells (ESCs), fibroblasts, partially or fully differenti- ated dopaminergic neurons, primary breast cancer cells (BCC) and For analysis of cell viability we used a colorimetric test based on leucocytes (Fig. 1A, Supplemental Fig. 1). Our hierarchical cluster tetrazolium salt — XTT (Roche Diagnostics, Indianapolis, IN, USA) analysis showed that genes encoding for CENPs are expressed in many according to instructions from the manufacturer and previously cell types and tissues (Fig. 1A). While the whole group of genes described procedure (Stangeland et al., 2015). P-values were encoding for CENPs seems to be universally down-regulated in calculated using non-parametric Mann–Whitney test. leucocytes the gene CENPC1 was up-regulated (Fig. 1A). Using Thesphereformingassaywasdonebyseedingsinglecell principal component analysis (PCA) we compared the expression suspensions containing 500 cells per well in ultra-low attachment flat profiles of genes encoding CENPs in the aforementioned set of cultures bottom 96-well plates for 10 days (Sarstedt). Subsequently, the plates and tissues. This analysis revealed that the majority of CENPs were imaged using GelCount™ (Oxford Optronics, Abington, UK). Only exhibited similar profiles (Supplemental Fig. 1A–B) except for spheres N50 μm were taken into consideration and 10 wells were CENPV, CENPF and CENPB that seem to be outliers (Supplemental evaluated for each cell culture. The number and the size (average area) Fig. 1A–B). Hierarchical cluster analysis further separated all samples of spheres were measured using software supplied by the manufac- into two categories (Fig. 1A). The first category consisted of turers. All results were presented as a mean of five to six independent undifferentiated and partially differentiated cell types while the experiments ± standard deviation. P-values were calculated using the second category contained predominantly differentiated cells such as non-parametric Mann–Whitney test. Sphere volume was calculated fully differentiated dopaminergic neurons and leukocytes (Fig. 1A). both by using the mathematical formula V = (4/3)πr3 (shown in Fig. 7 The “undifferentiated” category consisted of GICs, BCCs, GBM tissues, and the Supplemental Fig. 4) and confirmed using the sphere volume GBM commercial cell lines, ESCs and NSCs. When only this category function integrated in the Gelcount software (not shown). was analyzed the samples were separated roughly to NSCs and cancer stem/progenitor cell cultures (Fig. 1B). The “partially differentiated and 2.6. Immunolabeling mixed” cell cultures group consisted of undifferentiated and partially differentiated iPS, fibroblasts, some GBM primary cell cultures and a GIC cultures were plated in 24-well plates (Sarstedt) or chamber series of incompletely differentiated dopaminergic neurons (Fig. 1A). slides (Sigma-Aldrich) pretreated with Retronectin 50 μg/ml (Takara) By selecting the 17 most variable CENP-encoding genes in the GIC and incubated overnight to facilitate cell adhesion. Cells were fixed cultures and commercial GBM cell lines we separated these cultures with 4% PFA and washed with PBS. Immunolabeling was performed as into two clusters (Fig. 1C). Cultures in the first and the second cluster previously described (Stangeland et al., 2015). were characterized by relatively lowly and relatively highly expressed For detection of CENPA protein we used the primary antibody CENPV respectively. Interestingly, GBM commercial lines were low in Anti-CENPA [#2186, rabbit, 1:400, CST]. The secondary antibody was: CENPV. While the majority of the selected genes encoding CENPs were Alexa Fluor 594 A11012 (goat anti-rabbit, 1:500, Invitrogen). typically highly expressed in the selected cultures there were several cultures where the CENP genes were lowly expressed (Fig. 1C, red 2.7. Flow cytometry dendrogram branches/lines on the left). To further investigate the functional relationship between CENPA Spheres from GSC culture T65 were dissociated into single cells, and and other CENPs we downloaded gene expression data from the study incubated overnight to recover. Cells were pelleted, and incubated with where several hundreds of genes were knocked down (Fig. 1D and primary antibodies against CD133/2-PE conjugated (Miltenyi biotec) Supplemental Fig. 2A–B) (Hurley et al., 2012). This data set contained and SSEA1-PE conjugated (Becton-Dickinson). Cells were then ana- gene expression profiles of human umbilical vein endothelial cell lyzed using a LSRII flow cytometer (Becton-Dickinson). We used the (HUVEC) cultures featuring single knockdowns (KDs) of CENPA, following primary antibodies: Anti-Ki67 (ab16667, rabbit monoclonal, CENPB, CENPC1, CENPE, CENPF and CENPH. Global analysis of gene Abcam) and Anti-SOX2 (MAB2018, mouse monoclonal, RD Systems). expression revealed that knockdowns of CENPA and CENPF lead to similar gene expression profiles thus indicating a very close functional 3. Results relationship between these two CENPs (Fig. 1D). Furthermore, global gene expression profiles of cell cultures featuring knockdowns of 3.1. Expression of CENPA and other CENPs in GBMs, GICs, NSCs and CENPA, CENPF, CENPC1 and CENPB appeared to bear certain similarities variety of human cell types to one other (Supplemental Fig. 2A). Knockdowns of these four genes all down-regulated the same set of genes while the knockdown of Expression of CENPA and the genes encoding other centromeric CENPE and CENPH leads to the up-regulation of the same set proteins was assessed in a range of cell types, using our microarrays (Supplemental Fig. 2B). The set of genes, differentially regulated and public data available in the GEO repository (Fig. 1 and between the two groups, encompassed genes involved in regulation

Fig. 1. Expression of CENP genes in GICs and a variety of other cell cultures. A. Hierarchical cluster analysis of CENP gene expression performed on 134 microarrays separates predominantly undifferentiated (cluster 1) from partially differentiated (cluster 2) and differentiated (cluster 3) cell cultures. The majority of NSC and GIC cultures clustered with BCC and other undifferentiated cells. Cell cultures demonstrating stepwise differentiation from iPS to dopaminergic neurons in the course of 0–15 days clustered with iPS, fibroblasts, NFCs and a GBM tissue sample. Fully differentiated DA neurons (25 days of differentiation) clustered with other fully differentiated DA neurons and leukocytes. B. Expression of genes encoding CENPs in cluster 1. Hierarchical cluster analysis of cluster 1 from A revealed separation between normal undifferentiated cultures (encompassing NSCs and a few GICs) and undifferentiated cancer cell cultures (GICs, BCCs and a few normal NSCs and ESCs). C. Hierarchical cluster analysis of GIC cultures, GBM cell lines and three GBM tissue samples uncovered two clusters according to CENPV expression. This analysis also showed that several GIC cultures had low CENPs expression (red branches on the dendogram tree to the left). D. Global analysis of selected genes encoding CENPs in human umbilical vein endothelial cell (HUVEC) cultures featuring single gene knockdowns of CENPA, CENPF, CENPC1, CENPB, CENPE and CENPH. Using principal component analysis to compare gene expression profiles of the knockdown cultures to one another we uncovered similarity between CENPA and CENPF knockdowns. For more information on differentially regulated genes, 2D plots and statistical parameters for PCA see Supplemental Fig. 2. 10 J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18

AB C 10.0 ** CENPB Y 500 *** CENPB 400 300 5.0 CCENPTENPT CENPT 200 CENPC1 CENPQ Z 100 CENPC1CENPC1 CENPF CENPF 0 CENPA l CENPQ Expression units microarray units Expression a l 0.0 CENPM X m l ra CENPM sic y ra u CENPN s u e a e n CENPE

nch Component2 Principal cl e n o s r CENPA e p m CENPN Subtype CENPE -5.0 0.0 5.0 10.0 15.0 20.0 PC1: 58.11 % var. PC2: 19.6 % var. Principal Component1 Total variance retained: 77.9%

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Signature: 50 p=0.021 CENP(ANEFM) LOW Distance metrics: Pearson Correlation survival Percent CENP(BCTQ) HIGH Linkage: WPGMA 0 0 500 1000 1500 2000 -2 -1 0 1 2 Survival Days J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 11 of innate immunity, differentiation and mitochondrial function immunolabeling with a CENPA specific antibody (Fig. 3). CENPA (Supplemental Fig. 2B). protein was detected in the nuclei of GICs represented by a very specific spotty pattern (Fig. 3B). The intensity of the signal was 3.2. Expression of CENPs in clinical GBM samples from TCGA varying from nucleus to nucleus and some nuclei distinguished themselves by both very bright spotty CENPA staining and a “cloudy” The Cancer Genome Atlas (TCGA) database comprises of large appearance in the surrounding nucleoplasm (Fig. 3E). This additional amount of expressional data gathered from a vast number of cancer staining could indicate ectopic assemblage of CENPA protein in the tissues (http://cancergenome.nih.gov). The TCGA GBM tissue set was peri-centromeric regions or along the (neocentro- previously described and divided into four putative GBM subtypes: meres). CENPA was otherwise expressed exclusively in the nuclei proneural, neural, mesenchymal and classical according to the where it overlapped (Fig. 3C and F) with DAPI staining (Fig. 3A and D). expression patterns of 840 identifier genes (Verhaak et al., 2010). Expression of CENPA in GICs during was followed using – The GBM subtypes all carried distinct mutations and had a immunolabeling (Figs. 3 4). During interphase the expression of pronounced relevance in clinical settings. We analyzed the expression CENPA in GICs was nuclear and appeared typically either as spotty “ ” of CENPA in clinical GBM samples from the TCGA database. The patterns (Fig. 3B) or spotty + cloudy patterns (Fig. 3E). During expression of the CENPA gene was significantly higher in proneural prophase the spotty pattern disappeared and was replaced by a tumors than in mesenchymal and classical tumors (Fig. 2A). Further, diffuse appearance (Fig. 3H). During the other phases of mitosis the we analyzed the expression of all genes encoding CENPs that were typical patterns disappeared and the CENPA protein could also be present on the TCGA beadchip using PCA and cluster analysis. Global detected in the (Fig. 4B, E, H and K). The characteristic fi analysis of the expression data for 200 GBM tissue samples from TCGA mitotic gures were documented using DAPI staining (Fig. 4A, D, G (Verhaak et al., 2010) revealed similarities in expressions of CENPA, and J). CENPM, CENPN, CENPQ, CENPC1 and CENPT (Fig. 2B–C). PCA also revealed that PC1, PC2 and PC3 identified CENPF, CENPB and CENPE as 3.4. CENPA gene knockdown in GIC cultures outliers, respectively (Fig. 2B–C). We further tested if the genes encoding CENPs were differentially regulated in GBM subtypes using To investigate the function of CENPA in GICs we performed PCA. This analysis showed that the CENP gene expression patterns knockdown of this gene using RNAi technology. We utilized four clearly separated proneural and neural tumors from the mixture of different shRNA constructs and a non-silencing (NS) control. To classical, mesenchymal and few neural samples (Fig. 2D). Curiously, the assess the efficiency of CENPA gene knockdown we used qPCR unclassified TCGA GBM tissue samples (black) were not evenly spread (not shown), western blot (Fig. 5A–B) and immunolabeling (Fig. and congregated predominantly with the proneural or mesenchymal/ 6). Quantification of western blots showed that the levels of classical assemblies (Fig. 2D). CENPA were reduced by 20–70% in the knockdown cultures KD1, To analyze the co-expression of CENP genes we performed hierarchi- KD2, KD3 and KD4 (Fig. 5A–C). The relative expression of CENPA cal cluster analysis on the same set from TCGA (Verhaak et al., 2010). This protein in knockdown cultures was also investigated using analysis revealed consistently high expression of CENPF in clinical samples immunolabeling. This analysis showed that the levels of CENPA (Fig. 2E). CENPJ and CENPI were lowly expressed in GBM tissues and were were reduced in nuclei of knockdown cultures (Fig. 6). In therefore excluded from some analyses (Fig. 2E). The expression values of conclusion, the immunolabeling showed significant reduction in CENPA clustered together with CENPN, CENPE, CENPF and CENPM (Fig. 2E) CENPA protein levels in all four KD cultures (not shown for KD2). while CENPB, CENPI, CENPC1, CENPT, CENPQ and CENPJ clustered with one The intensity of CENPA-specific signal was lowest in KD3 and KD4 another (Fig. 2E). The two gene clusters: CENP(AEFNM) and CENP(BCTQ) (Fig. 6J and N). The GFP expression is presented in green (Fig. 6C, were confirmed using another clustering method (Fig. 2F). We further G, K and O). tested if the expression of the two following gene signatures: a) CENP(AEFNM)_HIGH/CENP(BCTQ)_LOW and b) CENP(AEFNM_LOW/ 3.5. Knockdown of CENPA reduces stemness and sphere forming ability CENP(BCTQ)_HIGH had an impact on patient survival. When all GBM of GICs tissue samples from the TCGA set (Verhaak et al., 2010)weretestedthere fi – was no signi cant effect on patient survival (Supplemental Fig. 3A B). Knockdown of CENPA gene resulted in impaired growth of GIC However, when only the mesenchymal tissue samples were analyzed (Fig. cultures grown as spheres (Figs. 5 and 7). GBM spheres featuring 2G) the expression of the signature CENP(AEFNM)_HIGH/CEN- CENPA KD were smaller in size (Fig. 5D and Supplemental Fig. 4A). To fi P(BCTQ)_LOW correlated with signi cantly worse survival (Fig. 2H). test the expression of markers for stemness and we performed flow cytometry analysis. This analysis revealed reduced 3.3. Expression of CENPA protein in GICs levels of the cell proliferation marker Ki67 and the stemness marker SOX2 in KD2, KD3, and KD4 cultures but not in the less efficient KD1 We have previously shown that CENPA was up-regulated in GIC culture (Fig. 5E). To investigate the degree of linear dependence cultures and GBM tissues at mRNA and protein levels (Stangeland et between the protein levels of CENPA and SOX2 and CENPA and Ki67 al., 2015). Here we evaluated the expression of CENPA in GICs using we compared the quantified western data to the FACS data using

Fig. 2. Expression of CENP genes in glioblastoma tissue samples from TCGA. A. TCGA divided all GBM clinical samples into four subtypes: proneural, classical, neural and mesenchymal (Verhaak et al., 2010). CENPA is significantly up-regulated in the proneural GBM subtype. Asterisks correspond to p values and indicate levels of significance. “**” here corresponds to p = 0.0043 while “***” corresponds to p = 0.002. P values were calculated using a Mann–Whitney non-parametric test. B. The expression of eleven CENPs in GBM samples from GBM TCGA. 2D plot of principal component analysis (PCA) showing separation of CENP genes according to their expression in GBM tissues from TCGA. PCA showed grouping of CENPs (CENPF, CENPB and CENPE were outliers). The higher proximity of CENP genes in PCA indicates a higher level of co-expression in GBM samples. C. 3D plot of the PCA analysis in B. D. PCA analysis of CENPs expression in subtypes of GBM from TCGA (same set as in A). This analysis showed that CENPs are not randomly expressed in GBM subtypes. The proneural (dark blue) and neural (light blue) subtypes could be separated from the rest of GBMs according to CENP expression. E. Hierarchical cluster analysis of CENP genes in GBMs from TCGA (Verhaak et al., 2010) with reference to the four subtypes. F. Hierarchical clustering with distance matrix analysis of the same set as in E. Analyses in E and F revealed two distinct CENP clusters: CENP-A, N, E, F, and M vs. CENP-B, I, C1, T, Q, and J. G. Expression of the nine most variably expressed CENPs in mesenchymal GBM samples. The survival of GBM patients indicated in blue (blue dendrogram branches) was plotted against the survival of patients indicated in red. H. The survival of patients was significantly different in the two groups as shown by a Kaplan–Meier (KM) graph (H). The blue signature [highly expressed CENP(AEFM) and lowly expressed CENP(BCTQ)] correlated with shorter patient survival. The red signature correlated with better survival. The p value was calculated using a log-rank (Mantel-Cox) test. For more information on KM graph of survival of all GBM patients from TCGA in relation to CENP expression see Supplemental Fig. 3. 12 J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18

Pearson product–moment correlation coefficient (PPMCCr) calcula- In one culture (KD1) we assessed the effect of CENPA KD on sphere tion. Correlation was excellent: r = 0.91 and r = 0.96 for CENPA/ forming ability also on days 42 (Supplemental Fig. 4C), 56 (not SOX2 and CENPA/Ki67 levels respectively. shown) and 73 (Supplemental Fig. 4D) after transduction (as outlined To assess cell growth of cell cultures featuring CENPA knockdown in Supplemental Fig. 4B). The parameters of the SFA in KD1 were we measured cell viability using XTT kit. Moderate but significant significantly reduced after 42 days in this culture and the difference reduction in cell viability was observed in all cultures (Fig. 7A). In the became even more prominent after 72 days (Supplemental Fig. 4C– KD1 cell culture cell viability was assessed in subsequent passages up D). Other cultures (KD2, KD3 and KD4) showed only small to 80 days after transduction (Supplemental Fig. 4B). This culture fluctuations after prolonged incubation. started showing reduced cell viability only gradually and after 72 days To calculate the degree of linear dependence between the in culture it was half as high as in the NS control (Supplemental Fig. knockdown efficiencies and the results of the functional assays we 4B). used again the Pearson product–moment correlation coefficient Sphere forming ability is usually considered as a retrospective (PPMCCr) calculation. Correlations between the average number of assay to estimate the number of tumor initiating cells. Spheres spheres and knockdown efficiency and the sphere diameter (median) featuring CENPA knockdown appeared smaller than the correspond- and knockdown efficiency were r = 0.67 and r = 0.81 respectively. ing “wild-type” spheres (Fig. 5D). To quantify the effect of CENPA KD Correlations between the average cell viability and knockdown on sphere forming ability we performed a sphere forming assay (SFA) efficiency and the average volume of spheres (per well) and (Fig. 7B–D and Supplemental Fig. 4C–D). SFA assays performed in all knockdown efficiency were r = 0.43 and r = 0.56 respectively. In four cultures showed reduced sphere forming abilities. While the conclusion, while the size and number of the spheres correlated nicely average diameter of spheres in NS was 94.93 ± 12.16 μm, the to the CENPA knockdown efficiency (at the protein level) the cell diameter of spheres featuring CENPA knockdown was reduced to growth parameters, such as cell viability and sphere volume (TVSPW) 81.26 ± 9.08 μm, 74.80 ± 10.18 μm, 80.88 ± 13.75 μmand showed moderate correlation. 86.50 ± 18.63 μm in KD1, KD2, KD3 and KD4 respectively (Fig. 7B). The average number of spheres in NS was 47.13 ± 8.72 (Fig. 7C). This 4. Discussion number was significantly reduced in the knockdown cultures: KD1 (27.97 ± 48.73), KD2 (26.80 ± 11.78), KD3 (25.07 ± 7.34) and KD4 Uncontrolled cell division is a hallmark of all cancers, including (28 ± 12.05) (Fig. 7C). The total sphere volume per well (TSVPW) glioblastoma. Consequently, several therapeutics that specifically was dramatically reduced in all tested knockdown cultures (Fig. 7D). target mitosis have been designed and tested in clinical settings While the average TSVPW in the NS culture was 2,261e7 ± (Dominguez-Brauer et al., 2015; Rath and Kozielski, 2012; De Witt 1,088e7 μm3, we calculated the average TSVPW of 8,656e6 ± Hamer et al., 2011; Herman et al., 2015; Kaneta and Ullrich, 2013; 5,150e6 μm3, 5,929e6 ± 2,909e6 μm3, and 7,448e6 ± 3,521e6 μm3, Lim et al., 2014; Sarcar et al., 2011; Chung et al., 2012). CENPA is a 1,075e7 ± 0,689e7 μm3 in KD1, KD2, KD3 and KD4 respectively (Fig. centromere-associated variant of histone H3 that plays an important 7D). role during cell division by ensuring proper separation of the sister

Fig. 3. Expression and localization of CENPA protein in GICs. A–F. Two cells showing typical (A–C) and strong (D–F) staining of CENPA. G–I. A typical prophase staining in GICs is shown. DAPI staining is shown in A, D and G. Immunolabeling with Anti-CENPA antibody is shown in B, E and H. Merge of the two is shown in C, F and I. J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 13 . It is frequently up-regulated in cancers (Zhang et al., Association of CENPA with stemness and highly proliferative state 2014; Habel et al., 2013; Toh et al., 2011; Rajput et al., 2011; Hu et al., was also observed in normal mammal cells (McGregor et al., 2014; 2010; Biermann et al., 2007a; Biermann et al., 2007b; Furukawa et Ambartsumyan et al., 2010). In cardiac progenitors CENPA is not only al., 2006; Li et al., 2007; Tomonaga et al., 2003; Qiu et al., 2013)and highly up-regulated but its expression is also increased in the embryonic has been suggested as a potential therapeutic target (Stangeland et heart and down-regulated in the adult heart and upon differentiation al., 2015; Li et al., 2011). A study in hepatocellular carcinoma (McGregor et al., 2014). Knockdown of CENPA in cardiac progenitors revealed that CENPA was highly up-regulated at mRNA and protein slowed down cell proliferation (McGregor et al., 2014). Our current levels in this cancer (Li et al., 2007; Li et al., 2011). Knockdown of study in GICs was in agreement with these results. We performed CENPA significantly decreased proliferation of hepatocellular carci- several tests to determine influence of CENPA knockdown on stemness noma cell lines and reduced tumorigenic features in vivo (Li et al., of GICs. The number of spheres retrospectively indicates the number of 2011). GICs in the culture. Our study shows that the knockdown of CENPA gene We have recently identified a CENPA containing co-expression severely impaired sphere formation by decreasing all three major module highly up-regulated in glioblastoma tissue samples and GICs parameters: number, diameter and volume of the spheres. However, (Stangeland et al., 2015). Among the nine genes, CENPA was the most only the number of spheres would suggest reduction in the population up-regulated one (Stangeland et al., 2015). Genes that show similar of GICs. The diameter and especially total volume of the spheres expression patterns might be functionally related. It was therefore not (TVSPW) are more related to cell growth (cell biomass) and so surprising that these genes encoded proteins that were all part of proliferative capability as we previously reported (Stangeland et al., the same functional network (Stangeland et al., 2015). 2015; Joel et al., 2015; Mughal et al., 2015).

Fig. 4. Expression and localization of CENPA protein in GICs during cell division. A–F. Typical metaphase staining. G–I. Typical staining in GICs is shown. J–L. Typical staining. DAPI staining is shown in A, D and G and J. Immunolabeling with Anti-CENPA antibody is shown in B, E, H and K. Merge of the two is shown in C, F, I and L. 14 J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 AC 1.2

1

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0 B D

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NON-SILENCINGCENPA KD1 CENPA KD2 CENPA KD3 CENPA KD4

5 5 5 5 5 10 10 10 10 10 87,7% 95,1% 54,2% 3,69% 19,3% 4 4 4 4 4 10 10 10 10 10

3 3 3 3 3 10 10 10 10 10 Ki67 2 2 2 2 2 10 10 10 10 10 0 0 0 0 0

2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10

5 5 5 5 5 10 10 10 10 10 79,2% 95,6% 44,2% 7,82% 38,2% 4 4 4 4 4 10 10 10 10 10

3 3 3 3 3 10 10 10 10 10 SOX2 2 2 2 2 2 10 10 10 10 10 0 0 0 0 0

2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10

Fig. 5. Knockdown of CENPA reduces proliferation and stemness of GICs. A–B. Confirmation of CENPA knockdown by western blot. Relative protein expression (RPE) values are indicated. C. Quantification of two independent western blots confirms good knockdown effect in several KD cultures. D. Growth and proliferation of GICs is reduced in CENPA knockdown cultures. Spheres of GIC culture transduced with non-silencing shRNA (left) were typically bigger than spheres of GIC cultures featuring CENPA KD (right). E. Flow analysis showed that the expression of the cell proliferation marker Ki67 and stemness marker SOX2 were both reduced in KD cultures. Error bars correspond to standard deviations.

In the present study we show that CENPA knockdown not only marker SOX2 in CENPA knockdown cultures. The decrease in CENPA reduced the stemness of the GICs but also the cell viability, although protein levels was also nicely paralleled by decrease in proliferation the effect was rather mild. The results of the functional essays (SFA marker Ki67 levels. This was in agreement with another knockdown and cell viability) were further confirmed by assessing SOX2 and Ki67 study where decrement in CENPA expression correlated with expression. We detected a decrease in the expression of the stem cell decreased Ki67 expression [56]. J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 15

Fig. 6. Confirmation of knockdown by immunolabeling. Typical CENPA staining in non-silencing (A–D), KD1 (E–H), KD3 (I–L) and KD4 (M–P) cultures. DAPI staining is shown in A, E, I and M. Immunolabeling with Anti-CENPA antibody is shown in B, F, J and N. GFP is shown in C, G, K and O. Merge of the two is shown in D, H, L and P.

Although the complete loss of CENPA led to genomic instability some cases CENPA can be detected in non-centromeric and embryo lethality in mouse (Howman et al., 2000) it has been regions where the protein might lead to changes in gene regulation shown that colorectal cancer cells can sustain some centromere and chromosome fragility (Lam et al., 2006). Furthermore it has been function and assembly of kinetochore proteins even with as little as shown that CENPA can be recruited by artificially induced DNA brakes 1% of residual CENPA protein (Fachinetti et al., 2013). Furthermore, in osteosarcoma studies (Zeitlin et al., 2009). This phenomenon seems the knockdown of CENPA in fibroblasts and iPS showed that the latter to be mediated by uracil DNA N-glycosylase (Zeitlin et al., 2011). Some, tolerated much better depletion of this gene (Ambartsumyan et al., so called, CENPA hotspots accumulate in chromosome telomeric regions 2010). While fibroblasts featuring CENPA knockdown could not such as region 8q24/Myc which is associated with genome instability sustain functional centromeric mark the iPS could (Ambartsumyan (Athwal et al., 2015). This particular region has been shown to influence et al., 2010). The abovementioned examples from the literature could glioma risk (Enciso-Mora et al., 2013). Our study showed that CENPA explain why the proliferation of our CENPA knockdown cultures was was especially highly up-regulated in proneural GBM samples. Each of only mildly affected in spite of very good CENPA knockdown efficiency the four GBM subtypes is defined with a specific set of expressed genes, and reduction in Ki67 levels. In conclusion, our study shows that CENPA genetic aberrations and epigenetic marks (Verhaak et al., 2010). It regulates stemness of GICs while the effect of this gene on cell proliferation is remains to be revealed why this particular subtype is associated with less evident. highest CENPA expression and if the eventual CENPA hotspot would be Although CENPA protein is typically affiliated with its involved in this. presence at active promoters has also been reported (Cole et al., A comprehensive bioinformatic analysis of CENPs as a group has 2011). Frequently, this CENPA is quickly targeted for degradation never been done. Neither has the role of CENPs in regulation of (Hewawasam et al., 2010; Ranjitkar, 2010). Abnormally increased stemness been determined. In our study we included a large set of CENPA expression, that is very often detected in human cancers, stem, progenitor and differentiated cells (Fig. 1). Although, we typically results in centromere expansion (Tomonaga et al., 2003). In focused especially on GICs this analysis revealed interesting 16 J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 A B CELL VIABILITY SPHERE DIAMETER 1.2

1.0 *** * **** **** 150 0.8 **** m 100 **** ***

0.6 µ Absorbace units 0.4 50 NS KD1 KD2 KD3 KD4 Diameter Cell culture 0 NS

CENPA CENPAKD1 KD2CENPA KD3CENPA KD4 C D NUMBER OF SPHERES SPHERE VOLUME

7 80 5 10

4 107 60 **** **** 7 m3 3 10 **** **** µ **** 40 **** 2 107 **** **** 20 1 107 Volume Number of spheres

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CENPA KD1 CENPA KD3CENPA KD4 CENPA KD2 CENPA CENPAKD1 KD2CENPA KD3CENPA KD4

Fig. 7. CENPA knockdown reduces cell viability and sphere forming ability of GICs. A. Cell viability in CENPA knockdown (KD) cultures was measured using XTT kit. Cell viability for NS and the KD cultures was calculated as an average of three passages. Cell passages were 4–6 after transduction. The values for each KD culture were significantly different from the NS control. Asterisks correspond to p values and indicate level of significance. In this case: pKD1 to NS = 0.0008; pKD2 to NS = 0.0206; pKD3 to NS b 0.0001; pKD4 to NS b 0.0001. The p values were calculated using a non-parametric Mann–Whitney test. Error bars correspond to standard deviations. B–D. Results of the sphere forming assay (SFA). The SFA parameters for NS and the KD cultures were calculated as an average of three experiments. Each experiment was conducted using three passages per culture. Cell passages were 4–6 after transduction. Each time 10 independent measurements (wells) per culture were conducted. B. Diameter of spheres was significantly reduced in KD1, KD2 and KD3 while in the KD4 was close to significance (p = 0.07). C. Number of spheres was significantly reduced in all four KD cultures. D. Volume of spheres was significantly reduced in all four KD cultures. Common for B–D: Asterisks correspond to p values and indicate levels of significance: * = (p ≈ 0.01–0.05), ** = (p ≈ 0.001–0.01), *** = (p ≈ 0.0001–0.001), **** = (p b 0.0001). The p values were calculated using a non-parametric Mann–Whitney test. Error bars correspond to standard deviations. information of CENPs' specific expression in differentiated cells and appropriately reflecting these functional groups. Out of 10 CENPs some tissues that we otherwise included only as controls (Fig. 1). For present on the bead chip (U/R/T/W/S and W were absent) nine were example CENPI and CENPH were relatively lowly expressed while grouped as in CCAN (underlined). Firstly, CENPC showed an none of the CENPs was universally expressed in all tested cell cultures. expression pattern quite different from the rest of the CENPs and CENPC1 and CENPV were specifically expressed in mature normal formed its own group (Fig. 1A). Secondly, L and N are in the same lymphocytes (Fig. 1A) and dopaminergic neurons (DNs), respectively. cluster while K, M and I clustered together. H clustered with L and N The latter was expressed both in DNs fully and those partially and was thus the only CENP outlier in this analysis. For all other CENPs differentiated from iPS and in a few GIC cultures. CENPF was highly the expression patterns at the mRNA level (hierarchical cluster up-regulated only in partially differentiated DNs and otherwise in analysis) reflected functionality in the CCAN complex. stem and progenitor cells, GBM, GICs and other breast cancer cells. All CENPs do not seem to be randomly expressed in subtypes of GBM other CENPs were predominantly up-regulated in undifferentiated tissues in TCGA (Fig. 2D). Proneural and neural groups exhibited CENP and partially differentiated cells and down-regulated in differentiated profiles quite distinct from the rest of tissues that belonged to classical cells. Further refinement revealed that among the undifferentiated and mesenchymal subgroups or were “unsorted” (black spheres in Fig. cells normal NSCs had slightly different profiles than the cancer cells 2D). Furthermore the two CENP signatures that we identified (Fig. 2F– and ESCs (Fig. 1B). The analysis of the entire CENP profile in a number G) correlated with very different patient survival (Fig. 2H). Worse of cell types also revealed that undifferentiated and differentiated survival correlated with highly expressed CENPs: A, E, F, N and M. Best cells have quite distinct CENP profiles (Fig. 1A). survival was obtained if these five CENPs were down-regulated Within the CCAN all CENPs can be divided in five groups (McKinley (although CENPs B, C, T and Q were up-regulated). Genes with similar and Cheeseman, 2016): 1) CENP(C), 2) CENP(LN), 3) CENP(HIKM), 4) expression patterns might be functionally related. In predominantly CENP(OPQUR) and 5) CENP(TWSX). The expression patterns of undifferentiated cells CENPA was co-expressed with CENPB, CENPN, selected CENPs in undifferentiated cells (Fig. 1B) seem to be CNEPL and CENPH (Fig. 1B). CENPN and CENPB are clearly very closely J. Behnan et al. / Neuroepigenetics 7 (2016) 6–18 17 functionally related with CENPA as they are both inner kinetochore Chen, J., Li, Y., Yu, T.S., McKay, R.M., Burns, D.K., Kernie, S.G., Parada, L.F., 2012. A restricted cell population propagates glioblastoma growth after chemotherapy. proteins (with CENPC) and directly recruited by CENPA (Guse et al., Nature 488 (7412), 522–526. 2011; Carroll et al., 2009; Ando et al., 2002). We previously showed Cheng, L., Wu, Q., Guryanova, O.A., Huang, Z., Huang, Q., Rich, J.N., Bao, S., 2011. Elevated that CENPF was highly up-regulated in GICs (Sandberg et al., 2013). invasive potential of glioblastoma stem cells. Biochem. Biophys. Res. Commun. 406 (4), 643–648. Both in GICs and in GBM tissues CENPA, CENPE and CENPF were the Chung, V., Heath, E.I., Schelman, W.R., Johnson, B.M., Kirby, L.C., Lynch, K.M., Botbyl, J.D., part of the same network (Sandberg et al., 2013; Stangeland et al., Lampkin, T.A., Holen, K.D., 2012. First-time-in-human study of GSK923295, a novel 2015). 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