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Targeting the Chromosomal Passenger Complex Subunit INCENP Induces Polyploidization, Apoptosis and Senescence in Neuroblastoma

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Targeting the Chromosomal Passenger Complex Subunit INCENP Induces Polyploidization, Apoptosis and Senescence in Neuroblastoma Author Manuscript Published OnlineFirst on August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0695 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Targeting the chromosomal passenger complex subunit INCENP induces 2 polyploidization, apoptosis and senescence in neuroblastoma 3 4 Ming Sun, Veronica Veschi#, Sukriti Bagchi, Man Xu, Arnulfo Mendoza, Zhihui Liu and 5 Carol J. Thiele,* 6 7 Cell and Molecular Biology Section, Pediatric Oncology Branch, Center for Cancer 8 Research, National Cancer Institute, Bethesda, MD 20892, USA 9 10 Running title: Targeting INCENP in neuroblastoma 11 12 * Corresponding author: Carol J. Thiele, Cell & Molecular Biology Section, 13 Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, 14 Bethesda, MD 20892. Phone: 240-858-3849; Fax: 301-451-7052; E-mail: 15 [email protected] 16 17 # Current address: Cellular and Molecular Pathophysiology Laboratory, Department of 18 Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, Italy 19 20 Key words: INCENP/CPC/Polyploidy/DNA damage/Apoptosis/Senescence 21 22 Conflict of interest 23 The authors declare no potential conflicts of interest. 24 25 26 27 28 29 30 31 32 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0695 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Abstract 2 Chromosomal passenger complex (CPC) has been demonstrated to be a potential target 3 of cancer therapy by inhibiting Aurora B or Survivin in different types of cancer including 4 neuroblastoma (NB). However, chemical inhibition of either Aurora B or Survivin doesn’t 5 target CPC specifically due to off-target effects or CPC independent activities of these 6 two components. In a previous chromatin-focused siRNA screen, we found that NB cells 7 were particularly vulnerable to loss of INCENP, a gene encoding a key scaffolding 8 component of the CPC. In this study, INCENP was highly expressed by NB cells and its 9 expression decreased following retinoic acid-induced NB differentiation. Elevated levels 10 of INCENP were significantly associated with poor prognosis in primary tumors of NB 11 patients with high-risk disease. Genetic silencing of INCENP reduced the growth of both 12 MYCN-wild-type (MYCN-WT) and MYCN-amplified (MYCN-amp) NB cell lines in vitro 13 and decreased the growth of NB xenografts in vivo with significant increases in murine 14 survival. Mechanistically, INCENP depletion suppressed NB cell growth by inducing 15 polyploidization, apoptosis and senescence. In most NB cell lines tested in vitro, 16 apoptosis was the primary cell fate after INCENP silencing due to induction of DNA 17 damage response and activation of the p53-p21 axis. These results confirm that CPC is a 18 therapeutic target in NB and targeting INCENP is a novel way to disrupt the activity of 19 CPC and inhibit tumor progression in NB. 20 21 2 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0695 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Introduction 2 Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. It 3 accounts for more than 7% of pediatric malignancies in patients under the age of 15 yet 4 causes 15% of all pediatric cancer deaths (1,2). NB is a neural crest-derived malignancy 5 and is presumed to arise from a failure of sympathoadrenal progenitor differentiation 6 during the normal development of sympathetic nervous system (3). During last few 7 decades, there has been significant progress in delineating the genetic underpinnings 8 involved in NB tumorigenesis leading to better risk stratification. This and the addition of 9 immunotherapy has contributed to the increased survival rate of NB patients (4-6). 10 However, overall survival of high-risk NB patients is still <50% despite intensive 11 multimodal treatment (2,6). Therefore, a better understanding of the functional 12 consequences of the genetic alterations in high-risk NB patients and a function-based 13 target selection strategy should lead to novel therapeutics. 14 15 In a chromatin-focused siRNA screen we identified 53 targets whose loss of function led 16 to decreased NB cell growth and/or increased NB differentiation. INCENP was identified 17 as one of those candidate genes whose silencing significantly inhibits NB cell proliferation 18 (7,8). INCENP encodes the Inner centromere protein (INCENP), which is the structural 19 and regulatory component of the chromosomal passenger complex (CPC) comprised of 20 INCENP, Survivin, Borealin, and the Aurora B kinase (9). CPC is responsible for proper 21 chromosomal alignment, segregation and cytokinesis during the mitosis (9). In the CPC, 22 INCENP plays two critical roles: firstly, it functions as a scaffold protein coordinating 23 assembly of this complex by interacting with all the other three components and 24 secondarily, the interaction between INCENP and Aurora B is necessary for activation of 25 the Aurora B kinase, the catalytic subunit of this complex (9). Thus, disruption of INCENP 26 expression leads to dissociation of the whole complex and limits Aurora B kinase activity 27 (10,11). Targeting the CPC has been led by strategies aimed at targeting Survivin or 3 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0695 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Aurora B kinase in NB since inhibition of either of them significantly blocks NB tumor cell 2 growth in vitro and xenograft growth in vivo (12-15). Recent Genome-Wide 3 Meta-Analyses (GWAS) studies have identified SNPs in INCENP that are linked to 4 increased susceptibility and risk in Breast, Ovarian, and Prostate Cancer (16,17). In 5 addition, INCENP has been found to be overexpressed in high grade non-Hodgkin B-cell 6 lymphomas and non-small-cell lung cancer, and proposed to be a biomarker for poor 7 prognosis in these types of cancer (18,19). However, the function and therapeutic 8 potential of targeting INCENP in NB remains unclear. 9 10 In this study we investigated how genetic targeting of INCENP affected NB cell growth. 11 We find that decreased INCENP levels are associated with increases in polyploidization, 12 apoptosis and senescence in vitro and decreased NB tumor growth in vivo. 13 14 Methods and Reagents 15 Cell culture 16 All the human NB cell lines used in this study were obtained from the cell line bank of the 17 Pediatric Oncology Branch of the National Cancer Institute and have been genetically 18 verified by short tandem repeat (STR) analysis and were routinely tested to be free of 19 mycoplasma. NB cells were routinely cultured in RPMI-1640 medium supplemented with 20 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 units/mL penicillin and 100 µg/mL 21 streptomycin (termed complete RPMI-1640 ). Stable shRNA NB cell lines were 22 maintained in complete RPMI-1640 containing 0.5 or 1µg/ml puromycin. Tet21N cells 23 were maintained in complete RPMI-1640 supplemented with 1μg/ml Tetracycline (Tet). 24 HeLa, 293T cells and ARPE-19 cells were cultured in DMEM with 10% FBS, 2mM 25 L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. For retinoic acid (RA) 26 treatment experiments, NB cells were treated with 5 μM of all trans-RA dissolved in 95% 27 ethanol and fresh RA containing medium was changed every 48 hrs. 4 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0695 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 2 Antibodies and Reagents 3 Primary antibodies against human INCENP (sc-376514), GAPDH (sc-25778), p53 (DO-I, 4 sc-126), MYCN (sc-53993) and Borealin (sc-376635) as well as HRP-labeled secondary 5 antibodies including Goat anti-mouse IgG-HRP (sc-2005) and Goat anti-Rabbit 6 IgG-HRP(sc-2004) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, 7 USA). The anti-MYC (5605S), anti-PARP (9532s), anti-Caspase 3 (9662s), anti-p21 8 (2947s), anti-Survivin (2808s), anti-pH2A.X (2577s), anti-pCHK2 (2197s), anti-Vinculin 9 (13901s), anti-Tubulin (3873s) and anti-Aurora B (3094s) antibodies were purchased 10 from Cell Signaling Technology (Danvers, MA, USA). Doxycycline (D9891) were 11 purchased from Sigma-Aldrich. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) 12 was purchased from Thermo Fisher Scientific. 13 14 INCENP silencing and rescue experiment 15 Transient INCENP knockdown was conducted by nucleofection or Lipofectamine® 16 transfection of different NB (except KCNR and Kelly cells) or control cell lines with two 17 different siRNAs targeting human INCENP (siINCENP-#2: 5'- GGATGGATCTGAATA 18 GCGA -3'; siINCENP-#4:5'- CCACGAUGCUGACUAAGAA-3') from Dharmacon, GE. For 19 INCENP knockdown in KCNR and Kelly cells, ON-TARGETplus pool siRNAs targeting 20 INCENP (Dharmacon, GE) were used. For MYCN knockdown, siMYCN (5'- CAGTATT 21 AGACTGGAAGTTCA-3', QIAGEN) was used. Nucleofection was performed in an Amaxa 22 Nucleofector II with Cell Line NucleofectorTM Kit V (VVCA-1003, Amaxa Biosystems, 23 Gaithersburg, MD, USA) according to the manufacturer’s instructions. A non-targeting 24 siRNA (siCTRL: 5'-UGGUUUACAUGU CGACUAA-3', Dharmacon) was used as a 25 negative control for gene knockdown experiments.
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