Published OnlineFirst December 12, 2013; DOI: 10.1158/0008-5472.CAN-13-1315 Cancer Molecular and Cellular Pathobiology Research Histone Chaperone CHAF1A Inhibits Differentiation and Promotes Aggressive Neuroblastoma Eveline Barbieri1, Katleen De Preter3, Mario Capasso4, Zaowen Chen1, Danielle M. Hsu2, Gian Paolo Tonini5, Steve Lefever3, John Hicks1, Rogier Versteeg7, Andrea Pession6, Frank Speleman3, Eugene S. Kim2, and Jason M. Shohet1 Abstract Neuroblastoma arises from the embryonal neural crest secondary to a block in differentiation. Long-term patient survival correlates inversely with the extent of differentiation, and treatment with retinoic acid or other prodifferentiation agents improves survival modestly. In this study, we show the histone chaperone and epigenetic regulator CHAF1A functions in maintaining the highly dedifferentiated state of this aggressive malignancy. CHAF1A is a subunit of the chromatin modifier chromatin assembly factor 1 and it regulates H3K9 trimethylation of key target genes regulating proliferation, survival, and differentiation. Elevated CHAF1A expression strongly correlated with poor prognosis. Conversely, CHAF1A loss-of-function was sufficient to drive neuronal differentiation in vitro and in vivo. Transcriptome analysis of cells lacking CHAF1A revealed repression of oncogenic signaling pathways and a normalization of glycolytic metabolism. Our findings demonstrate that CHAF1A restricts neural crest differentiation and contributes to the pathogenesis of high-risk neuroblastoma. Cancer Res; 74(3); 1–10. Ó2013 AACR. Introduction of residual disease of stage IV neuroblastoma after multimodal Neuroblastoma arises from residual immature neural crest therapy (3). Nevertheless, rising resistance and treatment tox- cells within the peripheral sympathetic ganglia of very young icity represent relevant limiting factors, and the overall children (1). The clinical and biologic behavior of this tumor is response rate to retinoic acid in patients with neuroblastoma fi extremely heterogeneous, encompassing fatal tumor progres- is low, suggesting that only a subgroup of patients bene ts from sion, as well as spontaneous regression and differentiation into the treatment. Therefore, a better understanding of the molec- mature ganglioneuroma. Furthermore, the degree of neuronal ular mechanisms that restrict neuroblastoma differentiation tumor differentiation strongly affects patient outcome. Studies could lead to improved therapeutic approaches for this highly from transgenic mouse models of neuroblastoma with targeted aggressive malignancy. overexpression of the MYCN oncogene also demonstrate that Alterations in components of the transcriptional machinery fi blocked neural crest differentiation leads to the malignant and chromatin modi er genes are now associated with initia- transformation of neuroectodermal precursors into neuroblas- tion and differentiation of multiple cancers (4), including neu- toma (2). Efforts to define the mechanisms for this blockage in roblastoma (5). A role for epigenetics in tumorigenesis is further neuroblast differentiation have been the focus of major research supported by recent genome-wide sequencing studies revealing efforts over the past years, and have led to the incorporation of recurrent cancer-associated mutations in key epigenetic regu- fi several differentiation strategies into neuroblastoma treatment. lator genes, including histone modi ers, histone chaperones, fi Retinoic acid therapy is an important component of treatment and DNA methylation modi ers (6). In particular, methylation of histone H3 at position lysine 9 (H3K9) has been extensively studied as a major factor regulating transition between tran- Authors' Affiliations: 1Texas Children's Cancer Center and Center for Cell and Gene Therapy; 2Department of Surgery, Baylor College of Medicine, scriptionally active euchromatin and inactive heterochromatin Houston, Texas; 3Center for Medical Genetics, Ghent University, Ghent, (7). In addition, H3K9 histone methyltransferases interact with 4 Belgium; CEINGE Biotecnologie Avanzate, Department of Biochemistry and DNA methyltransferases (e.g., DNMT1/3b) to indirectly modu- Medical Biotechnology, University of Naples Federico II, Naples; 5Pediatric Research Institute, University of Padua, Padua; 6Paediatric Oncology and late gene silencing through DNA methylation (8). The histone Haematology Unit "Lalla Seragnoli," Sant'Orsola-Malpighi Hospital, Univer- modifiers EZH2 (9) and LSD1 (10) are deregulated in neuro- sity ofBologna, Bologna,Italy; and 7DepartmentofOncogenomics,Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands blastoma with high expression conferring worse prognosis. In addition, repression of the tumor suppressor and chromatin Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). modi er CHD5 through loss of heterozygosity and DNA meth- ylation negatively correlates with long-term survival (11). Corresponding Author: Jason M. Shohet, Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Feigin CHAF1A (CAF p150) is a primary component of the chro- Building, Room 750.01, 1102 Bates Street, Houston, TX 77030. Phone: matin assembly factor 1 (CAF-1), composed of p150, p60, and 832-824-4735; Fax: 832-825-1604; E-mail: [email protected] p48 subunits, which promotes rapid assembly of nucleosomes doi: 10.1158/0008-5472.CAN-13-1315 on newly replicated DNA (12). The importance of CHAF1A in Ó2013 American Association for Cancer Research. cancer pathogenesis is supported by the finding that its www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst December 12, 2013; DOI: 10.1158/0008-5472.CAN-13-1315 Barbieri et al. overexpression has been linked to tumor progression (13), inter-run calibration, and error propagation) were performed cancer susceptibility (14), and more recently, epigenetic silenc- in qBasePlus version 1.1 (19, 20). ing (15). In addition, CHAF1A participates in a complex with MBD1 and SETDB1 during initiation of a gene-silencing pro- Short hairpin RNA constructs and antibodies gram by promoting H3K9 trimethylation, heterochromatin For p53 short hairpin RNA (shRNA), second-generation formation, and DNA methylation (16). lentiviruses expressing shp53 and shLuc control were used as We show here that CHAF1A restricts neuroblastoma differ- described (21). To knock down CHAF1A expression, a TRIPZ entiation using both in vitro and in vivo orthotopic models. lentiviral inducible shRNAmir with Tet-inducible promoter Elevated expression of CHAF1A indeed strongly correlates was used (Open Biosystems). The tetracycline response ele- clinically with an undifferentiated neuroblastoma phenotype ment (TRE) promoter also drives the expression of a TurboRFP and poor overall survival. We also demonstrate that CHAF1A reporter. To repress CHAF1A expression, doxycycline was promotes oncogenic signaling pathways (including RAS, AKT, added at a final concentration of 1 mg/mL. Control lines using BMI1, and WNT) as well as alters glycolytic metabolism path- scrambled shRNAmir were also generated. A GIPZ lentiviral ways. Together, these data support a novel function for the stable shRNA (Open Biosystems) was instead used to trans- histone modifier CHAF1A restricting neural crest differentia- duce neuroblastoma lines for in vivo studies. Briefly, 293T cells tion and promoting neuroblastoma tumorigenesis. were transfected with pLSLPw, TRIPZ, and GIPZ constructs along with packaging plasmids, pVSVG, and pLV-CMV-delta Patients and Methods 8.2 by using lipofectamine. Virus-containing supernatants Clinical patient cohort groups were collected at 48 and 72 hours and neuroblastoma cells Discovery set 1. Versteeg (n ¼ 88). This dataset of 88 unique were transduced in the presence of 8 mg/mL polybrene tumors was profiled on the Affymetrix HGU133 plus2.0 platform (Sigma). CHAF1A rabbit monoclonal antibodies (Epitomics; and normalized using the MASS5.0 algorithm. Expression data #5464-1; 1:500 dilution) and p53 mouse monoclonal antibodies were freely downloaded from the R2 website (http://r2.amc.nl). (Sigma; #P6874; 1:1,000 dilution) were used for Western blot- Validation set 2. Vermeulen (n ¼ 348). This cohort includ- ting. Anti-H3K9me3 antibodies (22-442; Millipore) were used at ed 348 patients with neuroblastoma taken from the Interna- a dilution 1:1,000 after acid extraction of the histones. tional Society of Pediatric Oncology, European Neuroblastoma Group (SIOPEN) and from the Gesellschaft fuer Paediatrische Xenograft model Onkologie und Haematologie (GPOH). Patients were only Orthotopic xenografts of human neuroblastoma were gen- included if primary untreated neuroblastoma tumor RNA (at erated as described previously (23) by injection under the renal 6 least 60% tumor cells and confirmed histologic diagnosis of capsule of an inoculum of 10 tumor cells in 0.1 mL of PBS. neuroblastoma) was available and of sufficient quality (17). Tumors were evaluated at necropsy 5 weeks after inoculation. Almost all patients were treated according to the SIOPEN protocols. The median follow-up was 63 months and greater Oligonucleotide microarray data analysis than 24 months for most patients (91%). In this cohort, 32% of Total RNA was isolated using the RNAeasy Kit (Quiagen) the patients had stage I disease, 18% stage II, 18% stage III, 23% from IMR32 cells transduced with inducible